Characterisation, antioxidant properties and cytoprotective investigation of Zanthoxylum zanthoxyloides root-bark in oxidative stressed promyelocytic leukaemia cells

Abstract

There is great interest in traditional or folk medicines in order to help with the discovery and development of potentially new pharmaceutics which have clinical benefits to man. Many of these traditional medicines are derived from medicinal plants which have been shown to contain a multitude of chemicals that have potent natural antioxidant properties. The chemical composition (metabolites), characterisation, antioxidant properties and cytoprotective effects to oxidative stress of Zanthoxylum zanthoxyloides (ZZ) root-bark extract obtained from different seasonal conditions, namely: winter, spring, summer and autumn bought from an African store and during one annual collection, were investigated in this thesis. ZZ root-bark was prepared in in two solvents (ethanol and water) and three methods of extraction (Aq decoction and maceration as well as ethanolic maceration). The extracts were investigated using different methods such as moisture content analysis, percentage insoluble fraction, spectrocolorimetric measurements, ash content analysis, analysis of carbon, sulphur, nitrogen, hydrogen and oxygen analysis, gas chromatography-mass spectroscopy (GC-MS), ultra-violet-visible spectroscopy (UV-VIS), Fourier-transform infra-red spectroscopy (FT-IR), qualitative and quantitative phytochemical analyses. Its antioxidative effects was investigated by assessing the plant extract’s ability to scavenge or react with a range of different free radicals such as: 2,2-diphenyl-1-picrylhydrazyl radical (DPPH), hydroxyl radical (●OH), singlet oxygen radical (1O2), nitric oxide radical (NO●), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS●+), metal ion, ferric reducing antioxidant power assay (FRAP) and reducing power assay. Lipopolysaccharide (LPS) and H2O2 were used to induce oxidative stress in human leukemic cells (U937 and HL60), and the ZZ extract was used to determine any potential cytoprotective effects to these oxidative insults. The result presented in this thesis showed that the moisture content of ZZ root-bark extract for winter, summer, spring and autumn was between 6.75 – 8.07% and this is within the acceptable moisture content percentage (5 – 8%). A statistically significant difference of *P<0.5 (winter vs spring), ***P<0.001 (spring vs summer) and ****P<0.0001 (winter vs summer) was observed among these seasons. The percentage insoluble fraction of this extract, in both solvents, was about 90% for these seasonal conditions with no statistically significant difference among them. The percentage ash content was between 6.9 – 8.3% for these harvest seasons, with statistically significant difference of *P<0.5 (winter vs spring) and **P<0.01 (winter vs summer), and this is within the acceptable range (5 – 40%). ICP-MS analysis of this extract confirms the presence of calcium, manganese, gallium, strontium, barium, lanthanum and cerium for all extracts from different seasons. GC-MS analysis of ZZ confirms the presence of many metabolites such as alkyl amide, lipids, coumarin, alkaloids, phenolics and triterpenes from all seasons. UV-VIS spectroscopy analysis further confirmed the presence of flavonoids for all seasons. Physico-chemical quantification analyses also confirmed the presence of many of these constituents in the extract. Percentage CHNS-O was between 43 – 52 % for carbon, 0 – 0.21 % for nitrogen, 4 – 12 % for hydrogen and 35 – 41 % for oxygen. However, sulphur was not present in all seasons. The ZZ extract also showed a great ability to scavenge free radicals such as DPPH● (IC50 = 30 μg/ml), ●OH (IC50 = 0.08 mg/ml), H2O2 (IC50 = 0.08 mg/ml), NO● (IC50 = 0.20 mg/ml) 1O2 (IC50 = 0.16 mg/ml) ABTS●+ (IC50 = 0.23 mg/ml) and metal ion (IC50 = 0.11 mg/ml) by accepting hydrogen or electron but showed a weak FRAP and reducing power ability. Finally, the extract showed it had cytoprotective effect against lipopolysaccharide and hydrogen peroxide-induced oxidative stress leading to cell death in U937 and HL60 leukemic cells. A more detailed characterisation showed that the ZZ extract was able to statistically protect against oxidative stress-induced cell death caused by either apoptosis or necrosis. In conclusion, ZZ extract possesses many phytochemical compounds, many of which have antioxidants properties regardless of their harvest seasons. Findings from this thesis confirmed the potential health benefits of ZZ root-bark and could be valuable for pharmaceutical and nutraceutical industries in using it to reveal new sources of natural antioxidant for therapeutic drugs.