Isolation and Characterisation of Quercitrin, a Potent Anti-Sickle Cell Anaemia Agent from the Nigerian Shrub, Alchornea spp

  • Olayemi Adedayo Adeniyi

Traethawd ymchwil myfyriwr: Traethawd Ymchwil DoethurolDoethur mewn Athroniaeth

Crynodeb

Alchornea spp. is an important plant genus in Africa, where it has been used traditionally by healers for the treatment of sickle cell anaemia (SCA). SCA is a genetic disorder endemic to African and sub-Saharan populations, along with other anaemic conditions. Plant leaves of this genus are used as a ‘blood tonic’ to reduce the symptoms of SCA in Nigeria, even though their active components or the mechanisms through which they act are not fully elucidated. In this research, the raw botanical, active botanical extract and purified active compounds of Alchornea spp. leaves were chemically characterized as a first step in the standardization of a phytomedicine and the development of an antisickling drug-lead. The project used a combination of various chromatographic and spectrometric techniques, including column chromatography, Liquid ChromatographyUltraviolet Mass spectroscopy (LC-UV/MS) to successively purify fractions, guided by bioactivities. Plant sample characteristics were assessed prior to characterisation of bioactive properties. Plant identifications linked to the Alchornea species was confirmed by DNA barcoding based on the rbcL gene sequence. Further phylogenetic assessments of plants sampled from various locations of Southwestern, Nigeria suggested no major genetic variation within the Nigerian samples. Flow infusion electrospray mass spectrometry (FIE-MS) for metabolomic profiling suggest variations in the metabolome which could reflect prevailing environmental factors as the coastal samples were distinctive. Pathway analysis suggested that the metabolomes differed in 5 metabolic pathways; biosynthesis of unsaturated fatty acids, glyoxylate and dicarboxylate metabolism, tricarboxylic acid cycle, ascorbate and aldarate metabolism, and ubiquinone and other terpenoid-quinone biosynthesis. This suggests stress factors such salinity along the coastal ecosystems could be influencing plant sample biochemistry. Results of studies directed at evaluating the anti-sickling activity of extract fractions/components and isolating the bioactive components present in the Nigerian Alchornea spp leaves, scientifically validated the in vitro potential of Alchornea spp in the management of sickle cell anaemia. Both the crude methanol extract and resultant fractions of Alchornea spp leaves demonstrated significant decrease in percentage sickling (>70% RBC sickling-inhibition) and rate of polymerization with increasing concentrations, prompting the isolation of components; ALM7T5 and ALM7V11. These compounds demonstrated significant anti-sickling activity, 87.16±2.39 and 93.06±2.69 % RBC sickling-inhibition at 0.4 mg/mL and 0.5 mg/mL respectively. To the best of our knowledge, this is the first time this property is being reported for this compound(s).Additionally, this work assessed Alchornea spp. extracts and fractions for their anti-oxidant properties. The results showed that the polar extracts of Alchornea spp. exhibited good antioxidant activities, with highest reducing power activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) at 66.40±1.59 % radical scavenging activity for methanol extract, followed by the aqueous extract at 61.71±2.45 % radical scavenging activity, at 400µg/mL concentration Structural Elucidation of ALM7T5 and ALM7V11, followed by further characterization identified these compounds as a single compound which both inhibits and marginally reverses sickling of sickled RBCs under hypoxic conditions, in vitro. The major anti-sickling chemical isolated from the Nigerian Alchornea spp., was identified by Nuclear Magnetic Resonance (NMR) to be quercitrin (quercetin 3-rhamnoside). In addition, the compound also inhibited Hb polymerisation possibly by increasing haemoglobin oxygen affinity. It also stabilized SCA-RBC membrane, possibly with increase in RBC deformability, and a reduction in RBC fragility. The impact of quercitrin on sickling was equally assessed using the metabolomics approach. Based on in-depth analysis of key metabolites and pathways, quercitrin-treated SCA RBCs metabolome depicted a metabolic signature similar to normal RBCs metabolome. This suggests that quercitrin could revert SCA RBC metabolomes to be equivalent to those of non-SCA RBCs. These findings are particularly exciting as they suggest that this compound may have multiple actions in reducing RBC sickling. This gives credence to the possibility of quercitrin mitigating vaso-occlusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo. The work described in this thesis has generated novel findings that are likely to inform efforts directed at the development of anti-sickling drugs.
Dyddiad Dyfarnu2020
Iaith wreiddiolSaesneg
Sefydliad Dyfarnu
  • Prifysgol Aberystwyth
GoruchwyliwrLuis Mur (Goruchwylydd) & Ifat Parveen Shah (Goruchwylydd)

Dyfynnu hyn

'