Ecballium elaterium (L.) A. Richard in Malta : the in vitro growth and quality of the Maltese squirting cucumber, a source of the potential anti-cancer tetracyclic triterpenoid, Cucurbitacin E.

Abstract

Ecballium elaterium L. is a local medicinal plant, containing elaterium, an extract rich in cucurbitacins (Cu), particularly cucurbitacin E (CuE). The aims of this dissertation were: 1. to determine the potential production of CuE in tissue culture. 2. to investigate further the effects of the compound on normal and malignant mammalian cell lines. A series of experiments correlating biomass accumulation and secondary metabolite production were carried out. Two experiments were carried out simultaneous to study the quality of calluses, the quantitative accumulation of cucurbitacins in tissue culture, and the effects of different PGRs on a particular callus strain. The best callus strain was strain 14 obtained from a seed excised from an immature fruit collected from M’Scala. The Cu and CuE contents correlated with the chlorophyll content of the calluses, indicating that a photosynthetic callus is capable of producing secondary metabolites. The PGR treatments of choice were NAA/BAP for maximum biomass accumulation and 2,4-D/Ki for the optimum cucurbitacin accumulation. The initial mixed PGR grid was carried out on strain 3 that was abundant at that time. Further experiments were carried out with strain 14. It was observed that when performing the above-mentioned 2-PGR grids, a growth-linked accumulation of secondary metabolites was observed. The lower metabolite yields with the 2,4-D/Ki combination prompted further investigations on the behaviour of callus in suspension with the above-mentioned treatments. It was observed that 2,4-D/Ki-treated calluses showed the greatest degree of metabolite leakage. To micro propagate plants from tissue culture, surface sterilised fruit were aseptically excised, the immature seeds taken and germinated on MS medium. The sterile plantlets obtained were excised and the cotyledons were cultured on MS medium containing 0.1 mg/L NAA and 1 ma/L BAP for four weeks, to enhance bud multiplication. Explants were then treated with various plant growth regulators (PGRs) or additives (i.e. IAA, IBA, NAA, 2,4-D, Ki, BAP, GA3, and charcoal). Shoots developed on most media but elongation was best observed with GA3. Some of the GA3-treated plantlets were treated for one week with IAA (auxin shock) that did not actually produce any effects on shoot, root or callus proliferation. With unsuccessful rooting, half of the plantlets were then treated with rooting hormone powder (NAA), and all planted in Jiffy pots and allowed to grow in a hot room. The plants that were given the auxin shock and then treated with rooting hormone showed the greatest mean apical height over a period of 75 days. The GA3-treated plantlets developed with greater difficulty especially the non- rooting hormone treated ones. From the pharmacological point of view, CuE was tested against five cell types; breast carcinoma (ZR-75-1), melanoma (COLO 679), prostate (PC-3), fibroblasts (L929) and peripheral lymphocytes, all obtained from the ECACC except for the peripheral lymphocytes that were obtained from healthy human male volunteers. The cultured cells were treated with different CuE concentrations. Specific control drugs were used for different cell lines. The total counts, viability, cytotoxicity, proliferation, cell morphology and apoptosis, were studied over a period of 72 h, in most cases. Control and untreated cells were assayed likewise. CuE exhibited a marked effect on PC-3 cells at a median inhibitory concentration (IC50) of 9.35 nM and moderate effects on COLO 679 and ZR-75-1 cells (IC50) = 87 and 1.95 uM, respectively). Parameters that showed a reduction in cell viability were prominent with the drug, as compared ‘o the controls. Morphologically, the cancer cells exhibited nuclear and cytoplasmic changes such as condensation of chromatin, an increase in the nuclear to cytoplasmic ratio, and rounding up of the cytoplasm. Surface blebbing and morphological signs of apoptosis occurred in all cancer cell types. In the agarose-gel electrophoresis analysis, DNA ladder characteristic of apoptosis, was exhibited by the CuE treatment on both PC-3 and ZR-75-1 cell lines, as for tamoxifen and mesterolone. Negligible cytotoxic effects were observed on the L929 cells and human T-lymphocytes as compared to the control. The fact that this compound did not damage human T lymphocytes but instead stimulated their activation and proliferation is a positive sign demonstrating that CuE is non- cytotoxic to normal cells and does not induce apoptotic changes seen with several other drugs. It was also observed that the compound is non-toxic even after the cells had been stimulated with PHA. The common parameter studied in the two different research fields, was CuE. The plant has been found to be a potential in vitro source for this compound and the latter has shown to be effective against certain cancer cell lines, not investigated or partly investigated previously. From the conclusions reached, research work could be directed towards bioreactor studies to optimise further, the yield of secondary metabolites in tissue culture, especially Cu and CuE. Also, animal trials, especially toxicological studies should be performed to determine the toxic effects of CuE on various organs and the immune system in vivo.