Danyaal, Mohammed (2020) The isolation and purification of chemical constituents of 'Croton megalocarpus' Hutch husks. (MSc(R) thesis), Kingston University, .
Abstract
Croton is a genus that is part of the Euphorbiaceae family. Croton is widely used in traditional medicine in a number of African countries such as Kenya, Tanzania, Uganda, Sudan, Mozambique and Zambia. Members of the Croton have been reported to treat malaria, diabetes, intestinal worms, influenza and arthritis along with other conditions. However, Croton megalocarpus Hutch is a species that is redominately found in Kenya. C. megalocarpus is regularly processed in the Croton factory that is situated in Kenya. The company that processes the nuts of C. megalocarpus is named Eco Fuels Kenya. The by-products from C. megalocarpus seed oil extraction includes fruit husks from the de-husking process and seed cake resulting from cold pressing of the oil. The purpose of this study is to see whether the husks of C. megalocarpus possess interesting compounds and to determine the safety of these compounds for alternative use. Chemical constituents found in the husks of C. megalocarpus, and various extracts obtained from the husks, seeds and fruit cake were tested for their cytotoxic effects against FM-55 human melanoma cells and mushroom tyrosinase inhibition effects. Extracts were prepared using hexane, dichloromethane and methanol solvents. The hexane extract of the husks produced an oil that had a similar profile to the cold pressed C. megalocarpus seed oil, the dichloromethane extract mainly exhibited diterpenoids of the labdane, cembrane and kaurane classes of compounds whereas methanol extract was mainly sugars with traces of magnoflorine and trans-4-hydroxyN-methyl-L-proline. The dichloromethane (DCM) extract was chosen for further analysis including additional extracts such as ethyl acetate and dichloromethane extract of Croton vinegar oil and tested for cytotoxic and anti- melanogeni9c properties. Seven compounds were successfully isolated from the dichloromethane extract of the husks of C. megalocarpus and characterised using nuclear magnetic resonance (NMR), high resolution mass spectrometry (HRMS) and infrared spectroscopy (IR). The seven isolated compounds are: trans ozic acid, sartone A, epoxykaurane, epoxychiromodine, vanillin, (-)-(1S*,4S*,10R*)-1,4-dihydroxycembra-2E,7E,11Z-trien20,10-olide and (+)-[1R*,4S*,10R*]-4-hydroxycembra-2E, 7E,11Z-trien-20,10-olide. In this study, the ethyl acetate extract of croton vinegar oil exhibited a tyrosinase inhibition of 59.33% at 20mM concentration. Epoxychiromodine exhibited an inhibition of 26.82% at 20µg/ml and showed to have a higher inhibition rate than the other isolated compounds. The dichloromethane extract of the husks was shown to have a cell viability of 45.62% (P<0.05) at 50µg/ml concentration. Sartone A was shown to have a cell viability of 86.08% (P<0.05) at 50µg/ml. The cytotoxicity and tyrosinase inhibition assay have helped determine the safety and effectiveness of the compounds and extracts that may play a vital role in skin care, skin lightening treatments and cancer treatment.
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