Investigation of a novel topical agent: the influence on the in-vivo biology of skin and the effect on bone healing (utilising critical defect and posterolateral fusion rabbit models, and fracture-fixation, epithelial hypertrophy and wound healing in rodent models)

摘要

Indigenous medical technology has a long clinical oral history in cultures around the world. The use of extracts from plants provides the foundation of many “therapies” around the world, while scientific examination of such treatments is lacking. The application of a novel, topical delivery derived from plant extracts that induced a physiological response to skin as well as bone is reported in this thesis. The novel preparation comprised of an extraction and concentration of particular plants that have had a long-standing but poorly understood use in Cook Island ethnobotany. The extraction procedure utilized edible plant oil derived from the coconut palm (Cocos nucifera), commonly referred to as coconut oil. This extraction technique was chosen based on the ethnobotany history of the Cook Islands and an understanding of transdermal pharmacology. To date, the scientific method has not been applied to this topical delivery of plant extracts nor has any identification of the potential active components been undertaken. This thesis outlines the work performed related to identification of the plants from the ethnobotany perspective, extraction technique and procedure, pre-clinical studies on skin and wound healing as well as bone repair and remodelling through fractures, defects and fusion. Finally, the thesis presents identification and analytical chemistry of the extracts. The plants used in this study are native to the Cook Islands and are identified in this thesis as A, N, P and K due to confidentiality. Of the four plant extracts A, N, P and K, in isolation the extracts of two of the plants (P and K) displayed activity, and a third plant (N) showed activity in high doses and in combination with the other two plants. Separations of the plants into solvent extractions showed the ability to concentrate and improved the biological effects by extracting plants into ethanol. However subsequent extractions into hydrocarbon soluble fractions showed biological activity but reducing complexity also reduced the biological effect. The presence of coconut oil as a control does not have a significant effect on the skin but it was essential for the biological effect of the plant extracts. The pre-clinical studies on skin examined the effects of the extracts themselves alone and in combination. Wound healing was also investigated as this has potential clinical significance. These investigation into the effects on the skin utilised 303 female Sprague Dawley rats and 34 NZ white rabbits. This work identified histological changes to the epithelium in the region of application of the plant extracts with no adverse reactions elsewhere at the tissue or organ level. A pilot study provided the initial assessment of the oil extracts on the skin of rats demonstrating a biological response. Further studies were designed based on the pilot study considering different plant components and chemical procedures and effects on the skin. The animals tolerated treatment well in the skin experiments. There were no deaths or illness associated with the topical treatments. The region of skin applied did not appear inflamed macroscopically or microscopically in any active treatments. The difference between the regions of application and control regions revealed a change to the epithelial structure with hypertrophy of many layers of the epithelium and increasing the stratified appearance. The increase in thickness was typically over 100% above the adjacent regions of the same animal, and higher if compared to untreated animals. This was reproducible and statistically quantifiable and may have implications for clinical application ranging from disorders of keratinisation, skin regeneration and repair through to age related atrophy.The effect seen on the epithelium of the skin was the same in young animals as it was in aged animals and even in aged oestrogen deficient animals. This is despite the differences in the baseline histology of the untreated animals in each of these groups. The effect of the extracts on the skin of rabbits was similar to that seen in the rats with changes to the epithelium seen and the epithelial structures such as hair follicles. The plant extracts in coconut oil were found to have a non-significant trend towards increasing mechanical wound strength when applied to a surgical wound and histological assessment of the wounds suggested a more rapid healing rate. The investigation into bone healing of the plant extracts incorporated three established models of fracture healing, defect healing and spinal fusion. These models represent typical pre-clinical scenarios for the evaluation of treatments to improve or augment bone healing. The plants extracts developed in the chemical phase of this thesis were applied topically in every case to the skin of the upper back and not applied to the surgical sites. Endpoints included radiographic, computed tomography, mechanical testing, histology and immunohistochemistry to examine the effects at different levels from the tissue to the cell. Radiologically, there was some evidence of increased bone formation in the treated groups. Histological profiles of the treated regions of bone healing as compared to the controls suggests an increase in vascularity, a decrease in macrophage inflammatory response, an increase in cartilage formation and an increase in new bone.It is apparent that it is absorbed transdermally, and requires coconut oil for this to occur, or coconut oil may play a role in metabolism of the agent. The effect is on regions of bone distal to the site of application and therefore appears to be a systemic treatment with a local response. The process by which this occurs from a systemic approach is still unclear. The response in treated animals from the first week onwards which when compared to control animals was evident and consistent. The results were reproducible in repeat experiments and were enhanced by concentrating the extracts. The response was evident in ovarectomized as well as in sham rats. Due to the small numbers, there was no means of achieving statistical significance. The pattern of increased cartilage and new bone formation was present in autograft spinal fusion at 6 weeks and at 8 weeks, with the new bone and cartilage forming fused mass.Mechanical testing was performed on spinal fusion mass and compared favourably to control autograft, with no statistical difference between the 8-week treated and the 12-week controls. High Performance Liquid-Gas Chromatography, UV Spectroscopy and Mass Spectrometry were performed on a fraction of the active separations. There is a signature peak differentiating the active sample from the control at the same point on Mass Spectrometry and UV Spectroscopy however this peak could not be identified in analysis of the plant extracts independent of the coconut oil.The “active” component was extractable by ethanol from Plant A and remained active in the absence of the other plants that did not produce an effect on bone when tested independent of plant A. The active fraction of plant A was found to be soluble in ethanol and hexane and required coconut oil for topical delivery to be effective. Chemical analysis of plant A extracts allow some insight into possibly active components which will need further investigation, and potentially a possible mechanism by which these observations are being mediated. In summary, this thesis presents a journey of discovery and understanding in the modern era of what has been “known” by the indigenous healers of the Cook Islands. The understanding of the biological effects as well as potential new clinical applications may provide new treatment strategies for the future.