Separation and characterization of saponins from the bark extract of the South American soap bark tree; Quillaja saponaria Molina: (potential immuno-adjuvant active compounds)

Tebogo, Motshegwana Olenkie (2004) Separation and characterization of saponins from the bark extract of the South American soap bark tree; Quillaja saponaria Molina: (potential immuno-adjuvant active compounds). Masters thesis, Memorial University of Newfoundland.

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Abstract

In recent years, researchers have demonstrated that the saponins derived from the bark extract of the South American tree, Quillaja saponaria Molina, possess an exceptionally potent immune stimulating capability. This has kindled much interest in their potential development as immune-adjuvants. These saponins have also been shown to play a very important role in the formation of immune stimulating complexes (ISCOMs), which are a specific type of antigen/adjuvant delivery system consisting of a cage-like structure made up of a Quillaja saponin, cholesterol and phospholipids in a 1:1:1 ratio. The purified fraction of the bark extract, known as Quil-A, has been shown to contain more than 60 saponin compounds. The structures of several of these have been reported, while some have only been partially characterized. While the use of Quil-A as an adjuvant is well established in veterinary medicine, its use in humans has not been achieved due to its toxicological effects and the fact that its composition cannot be standardized from batch to batch. Adjuvant research has become very important for the successful development of modern subunit vaccines as these vaccines do not elicit an immune response sufficient to grant an adequate protection to the subjects when administered alone. -- In this research saponin compounds from Qui I-A, a commercially available bark extract of Quillaja saponaria Molina, were separated, purified and structurally characterized. The biological activity of the individual compounds or fractions isolated to near purity have also been investigated. Compounds characterized that had novel structures were screened for possible adjuvant properties and compared with other compounds of known structures. -- Preliminary High Performance Liquid Chromatography (HPLC) separation of the extract Qui I-A by gradient elution using an ammonium acetate (10mM)/acetonitrile/water solvent system on C5 reverse phase high performance liquid chromatography (RP-HPLC) column has resulted in the purification of a saponin compound having a molecular weight of 1560 m/z. Mass spectrometric analyses, which included utilization of techniques such as collision induced dissociation and tandem mass spectrometry, afforded a proposal of the molecular structure and fragmentation pattern of this compound designated QF-23. Large scale separation of the material Quil-A over fifty HPLC runs has enabled the acquisition of five more fractions in pure or near pure form, including two that are documented to have been previously isolated by other researchers, as well as a sufficient amount of pure compound QF-23 to facilitate the performance of nuclear magnetic resonance experiments (NMR) to confirm the structure of this compound. Liquid Chromatography/Electrospray Ionization-Mass Spectrometry (LC/ESI-MS) has been employed to investigate further and more specifically the elution characteristics and composition of the crude material QuiI-A. The results of this experiment has shown Qui I-A to be a highly complex mixture of saponins and nonsaponin components which have a tendency to co-elute. Over 80 HPLC peaks were detectable by means of a photodiode array detector. Solid phase extraction technique was capable of eliminating some materials from Quil-A. -- Biological investigation of some saponin fractions revealed a fraction that had considerably lower in vitro toxicity to mouse monocytes than the other fractions tested, while the toxicity of QF-23 was found to be moderate and comparable to that of the well-established QS-21. In vitro studies have shown the compound QF-23 to be capable of stimulating mouse monocytic cells to produce the cytokine IL-1 alpha, thus suggesting that it may have immune-adjuvant properties.

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