Lipid Raft Biodirected Screening And Mechanistic Action Of Antitumor Lead Compounds From Arisaema Erubescens (Wall) Schott And Vaccaria Segetalis (Neck)

Tropomyosin -related tyrosine kinase A (TrkA) receptors are highly expressed in some cells like glioma, prostate, pancreatic and breast cancer cells. These receptors could serve as important cancer biomakers. The cancer biomarkers provide tools for risk assessment, diagnosis, prognosis and monitoring of the disease. In research, they are employed as drug target development and surrogate end points. This study employed TrkA receptor-rich U251 glioma cells as potential drug target. Here, lipid raft biomaterial with abundant TrkA receptor was developed from U251 cells and activated silica beads to direct online isolation of antitumor agents. It is worth noting that the majority of cancer patients have limited treatment options due to low therapeutic efficacies, severe side effects, high cost of drugs and increased resistance. The prevailing conditions call for new research directions to identify novel lead compounds to complement and enhance existing antitumor agents. Currently, about 60% of cancer drugs in clinical use could be traced to natural-based products, and the plant kingdom has been the most predominant source. However, inadequate bioscreening systems, numerous multi-components and unclear mechanistic actions of these products, coupled with lack of quality control measures have greatly hampered the efficacy of these promising agents. It is therefore refreshing when new biomedical technology and emerging plant sources are discovered to provide the needed support.With paradigm shift from single to multitarget drug discovery, indigenous herbs could serve as potential sources for unearthing multitarget antitumor lead compounds. The current bioscreening systems require dramatic changes to discover more antitumor agents which act on multiple targets. This study was therefore aimed at isolating antitumor components from two lipid raft-screened Chinese herbs, [{Arisaema erubescens (Wall) Scott, Chinese name-tiannaming, English name-Jack-in-the-pulpit; Vaccaria segetalis (Neck), Chinese name-Wangbuliuxing, English name-Cowherb/Cow cockle], which could account for the reported tumour growth inhibitory activities of the plants. The herbs are widely distributed in China and readily available on the market. Traditionally, the tubers of A. erubescens are used as analgesic, antitumor and pesticide agents, while the seeds of V. segetalis are used to activate blood circulation, treat amenorrhea and mastitis, promote milk secretion, heal wounds, and even serve as diuretic, astringent and anti-cancer agents. Phytochemical studies have shown that the A. erubescens contains alkaloids, saponins and triterpenoids. Similar reports have also revealed the main components of V. segetalis as flavonoid glycosides, cyclic peptides, triterpenoid saponins, lipoids, fatty acids, and monosaccharides. This study therefore reports lipid raft bioguided isolation, optimazation and mechanistic actions of antitumor lead compounds from two potential Chinese herbs. There are five chapters in this thesis with four major areas of contributions as detailed below:1. Natural product as an effective source of novel antitumor lead compounds.This section provides an extensive insight into the successful story of natural products, specifically Chinese herbal medicine (CHM), as a repository of bioactive compounds for cancer therapy. The blue print for isolating and identifying these unique compounds are highlighted in this report. Recent screening techniques that have supported the discovery of potential antitumor leads are also emphasized. The need to actively explore the effective parts of natural products, especially at the extract and formulation levels, for its potential multitarget antitumor activities are further showcased. In appreciating the situation, two natural heuristic Chinese herbal products (V. segetalis and A. erubescens with outstanding ethnomedicinal, phytochemical and pharmacological properties as screened via lipid raft silica bead biomaterial were used as a case study to give credence to the above information. The literature therefore provides a solid foundation for the development of subsequent work in this study.2. Lipid raft bioassay-directed screening for potential antitumor medicinal herbs.Here, ten (10) selected Chinese herbs, traditionally used to treat symptoms consistent with cancer, were rapidly screened with lipid raft affinity system. The process involved the preparation of lipid raft silica beads (LRSB) with sufficiently expressed TrkA receptors.The LRSB was then packed into a column to serve as an online stationary phase to screen different Chinese herbs for antitumor activities. Two potential antitumor herbs, A. erubescens and V. segetalis, were identified via the LRSB with chromatographs exhibiting long characteristic retentions after aqueous extract bioanalysis. The active herbs were separated into various fractions (petroleum ether, ethyl acetate, n-butanol and raffinate extracts) for further bioscreening. Conventional MTT bioassay was employed to authenticate the susceptibility of liver cancer cells to the final bioactive product of the herbs. Cytotoxic activities were observed in n-butanol extracts of the two identified herbs. The lipid raft screening report was also in agreement with MTT bioassay analysis. Consequenetly, the n- butanol extract for each bioactive herb provided the baseline reference material for subsequent separation, identification and mechanistic action of antitumor lead compounds.3. Isolation and identification of antitumor effective componentsIn this chapter, cytotoxic lead compounds which could be responsible for the reported antitumor activities of A. erubescens and V. segetalis were separated and duly characterized. The bioguided isolation and identification were performed at three major stages:multiple extractions, fractionations and purification. Structures of bioactive compounds were determined using the 1H,13C nuclear magnetic resonance data, electron ionization mass spectrometry and literature information. Some prominent cytotoxic compounds including vaccaroside E, segetoside H, segetoside I and other new agents with molecular weights of NC913, NC1482 and NC1776 were isolated from V. segetalis. Generally, the bisdesmosidic saponins were the most significant cytotoxic compounds in the seeds of V. segetalis as reported in previous studies; however, certain cyclopeptides in this study revealed similar activities. The most active compounds (segetoside I, NC913, NC1482 and NC1776) showed broad spectrum cytotoxic activities against several human cancer cell lines (HepG2, liver; U251, gliocoma; BGC, HGC & SGC, gastric; Lovo-1 Colon), with purity of between 75% to 85%. The activities of these compounds, particularly the bisdesmosidic saponins could be related to the presence of more sugar moieties and the different types of sugar units. The effectective components of A. erubescence also showed significant cytotoxicity against HepG2 cells which was to some extent comparable to the 5-Fluorouracil (5-FU). Further purification is needed to unearth significant lead compounds from this medicinal plant.4. Optimization of yield of cytotoxic compounds from Vaccaria segetalisIn this report, bisdesmosidic saponins showed the most significant cytotoxic activities among the bioactive isolates of V. segetalis. Unfortunately, such products are usually obtained in minute quantities which limit the comprehensive preclinical and clinical investigations in drug development process. The establishment of an effective extraction model is therefore required to enhance the yield of the lead compounds for novel cancer drug discovery. Based on our preliminary and other related studies which singled out microwave-assisted extraction (MAE) as the best extraction method as compared to other alternatives (Maceration extraction, ME; Ultrasonic-assisted extraction; UAE; Heat reflux extraction, HRE), the MAE was selected for optimization studies. The results revealed that the different alternatives did not significantly affect the quality of isolated compounds. The optimized experimental conditions of MAE led to 50% ethanol concentration,40 mL/g liquid/solid ratio,25 min time, 140 W microwave power and 0.765% extraction yield of cytototoxic bisdesmosidc saponins (CBS), while economically the most acceptable conditions gave 40% ethanol concentration, 30 mL/g liquid/solid ratio,10 min time,100 W microwave power and 0.729% extraction yield. The characterized CBS containing some prominent compounds like vaccaroside E-1422, segetoside H-1448 and segetoside I-1464 exhibited significant in vitro cytotoxic activities against HepG2 liver and SCG gastric cancer cells. The cytotoxicity of CBS was comparatively better than the clinically used drug,5-FU. These findings indicated that the MAE was the most efficient technique for CBS isolation, and the significant cytotoxic activities of bisdesmosidic saponins supported the cancer-related health benefits associated with V. segetalis.5. Basic mechanistic studies on segetoside I, a plant-derived bisdesmosidic saponin from Vaccaria segetalis.The study was aimed at ascertaining the inhibitory effect and possible mechanistic action of segetoside I on the viability of different cancer cell lines such as HepG2, U251, BGC, HGC, SGC and lovo-1. Here, the cancer cells were treated with different concentrations of segetoside I and the cell viability evaluated via MTT bioassay. The in vitro apoptotic inducing effect of segetoside I on the cancer cells was investigated via morphological analysis of Hoechst and AnnexinV/Propidium iodide staining, flow cytometry assays, cell migration and deoxyribonucleic acid (DNA) fragmentation. The in vivo growth inhibitory activities of segetoside I was also assessed in HepG2 xenograft tumour mouse. The MTT bioassay analysis showed that HepG2 cells were the most susceptiblewith lower toxicity in healthy mouse embryonic fibroblast cells. Segetoside Ⅰ-pretreated HepG2 cells further led to early apoptosis, dose-dependent DNA fragmentation, inhibition of cell migration, up-regulation of Bax (Bcl2-assoaciated X protein) and down-regulation of Bcl-2 (B-cell lymphoma 2), which indicated that an apoptotic signalling event could have been initiated. The segetoside I suppressed hepatoma growth in mice with virtually no toxicity, making it a strong oncology drug candidate. These data revealed that segetoside I could exhibit its antitumor activity via apoptotic induction and further support the possible application of this antitumor agent as a potential chemotherapeutic candidate worthy of future investigations.