Madala, N. E.Tavengwa, N. T.Ndlala, A. R.Ndou, Dakalo Lorraine2024-10-232024-10-232024-09-06Ndou, D.L. 2024. Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools. . .https://univendspace.univen.ac.za/handle/11602/2769Ph. D. (Chemistry)Department of ChemistryMoringa oleifera is a tree that has been studied extensively and it has been found to host a variety of medicinal and nutritional properties. Owing to these properties, it has often been referred to as a ‘miracle tree’. M. oleifera contains a variety of metabolites such as flavonoids, glucosinolates, phenolic acids, tannins, and carotenoids that are responsible for the pharmacological properties of this plant. In this study, the metabolite of interest in M. oleifera was the rutin flavonoid. Rutin has various pharmacological properties and its presence in M. oleifera makes the plant more bioavailable. In this study, the presence of rutin was evaluated in M. oleifera plants in households from different villages within the Vhembe District. The Molecular Networking (MN) approach was utilized to revisit the chemical space of flavonoids in M. oleifera. The aim was to establish the biochemical modifications responsible for the chemical diversity of these compounds, which have been reported to be associated with the purported pharmacological properties of this plant. Modern extraction methods such as ultrasonic-assisted aqueous two-phase extraction (UA-ATPE) and pipette tip micro-solid phase extraction (PT-μSPE) were explored in the extraction of the most bio-available and most sought-after flavonoid, rutin, in M. oleifera leaf extracts. This work has been divided into four experimental chapters. In Experimental Chapter 4, the presence of rutin was evaluated in the leaves of 135 M. oleifera plants from households in different villages within the Vhembe District of Limpopo Province of South Africa. The metabolite extraction was carried out using the conventional liquid extraction method using 80% MeOH and the resulting extracts were analyzed using UHPLC-qTOF-MS. The results from the UHPLC-qTOF-MS showed that only 15 plants produced rutin. This was confirmed using tandem mass spectrometry (MS2) and an authentic standard, which further validated the detected ion as a true representation of rutin. It was concluded that different cultivars of this plant are being grown in various households within the Vhembe District. These differences are expected to result in a negative perception towards Moringa plants, and as such, knowledge of the cultivar-chemical relationship should be made public with the intention to encourage the cultivation of proper plant species. The extraction of rutin from M. oleifera leaves using UA-ATPE was reviewed in Experimental Chapter 5. An ethanol/salt ATPE was formed for the extraction of rutin. Ultrasonication was used to assist in the extraction of rutin from the leaves. Central composite design (CCD) was used to design experiments and two factors which were optimized are ultrasonic time and ultrasonic temperature. The ammonium sulphate ((NH4)2SO4), sodium chloride (NaCl), and magnesium sulphate (MgSO4) salts were used to form the ethanol/salt ATPE two phase system. The resulting Response Surface Model (RSM) was observed to be a linear fit for the ethanol/(NH4)2SO4 and the ethanol/MgSO4 ATPE systems, with R2 values of 0.7339 and 0.5782, respectively, as obtained from the analysis of variance (ANOVA). The ethanol/NaCl ATPE system yielded a quadratic fit with R2 = 0.7865 and was observed to be the best performing in the extraction of rutin from the M. oleifera leaves with optimum extraction at a temperature of 25 ºC and time of 22.5 minutes. Based on multiple reaction monitoring (MRM) through the UHPLC-qTOF-MS technique, the concentration of rutin extracted by the ethanol/NaCl ATPE system was 240 μg L-1. According to ANOVA, temperature (the B-term) was found to be the significant term with a p < 0.0500 that the extraction of rutin through UA-ATPE is temperature-dependent. Therefore, it was concluded that the extraction of rutin from M. oleifera leaves favors low temperatures. The results of the current study further demonstrate the usefulness of simple extraction techniques, such as heated water with additives like salts, as a feasible method to enrich pharmacologically relevant metabolites from plants. This reaffirms traditional protocols that are currently used by communities which include boiling plants in water to extract useful chemical compounds for the treatment of common ailments such as colds and headaches. In Experimental Chapter 6, PT-μSPE was applied in the extraction of rutin from M. oleifera leaves using activated hollow carbon nanospheres (HCNSs) as the sorbent. The activated HCNSs were characterized using FTIR, which confirmed the presence of the functional groups of interest such as OH stretch, -COO- vibration, and C=O stretch. TGA thermogram showed a difference in the thermal stability of the raw and activated HCNSs, proving that the raw HCNSs are more stable than the activated HCNSs, and SEM displayed the difference in the morphology of the raw and activated HCNSs by observing the difference in the coalescence of the material. Parameters such as loading cycles, elution solvent, concentration of rutin, pH, loading volume, and mass of sorbent were optimized. The analysis of the extracts was conducted with a UV-Vis spectrophotometer to ascertain the recovery of rutin. The optimal conditions for rutin recovery using PT-μSPE were determined to be 15 loading cycles, i-PrOH as the elution solvent, a 2 ppm as standard concentration of rutin, a pH of 2, 500 μL as loading volume, and 1.5 mg of sorbent. The LOD, LOQ, and RSD values were found to be 0.604 mg L-1, 1.830 mg L-1, and 3.26%; respectively. It was thus confirmed that the PT-μSPE method is effective in the extraction of rutin even at trace levels based on the low LOD value obtained and the RSD value obtained proved that this method is a reliable pre-concentration technique and is thus repeatable for the analysis of complex samples. Therefore, in cases where some plants produce these compounds in minute concentrations, methods such as this one presented herein can be used to estimate and concentrate the pharmacologically relevant compounds. Molecular networking (MN) was used to study the global metabolic profile of M. oleifera and is outlined in Experimental Chapter 7. Herein, Global Natural Products Social (GNPS) platform was used to generate the MN from the LC-MS data obtained from the methanolic leaf extracts of M. oleifera. The MN was viewed and analyzed using Cytoscape. Through MN, it was observed that M. oleifera contains a variety of metabolites. Other GNPS tools such as network annotation propagation (NAP), DEREPLICATOR, MS2LDA, and MolNetEhancer were further used to compliment the classical MN model. To this end, MS2LDA was used to annotate the flavonoids found within M. oleifera. Kaempferol, quercetin, and isorhamnetin flavonoids were successfully annotated by MS2LDA. Additionally, Chrysin-6,8-C-diglucoside was also annotated and reported for the first time in M. oleifera leaves. The results of this study further suggest MN models as useful tools for chemical exploration, enabling the discovery of new metabolites by leveraging existing knowledge as "chemical charms" to unearth hidden metabolites.1 online resource (xix, 198 leaves) : color illustrationsenUniversity of VendaUCTD583.640968257RutinAlcoholsBioflavonoidsMoringa -- South Africa -- LimpopoMoringa olieifera -- South Africa -- LimpopoMoringaceae --South Africa -- LimpopoPapaverales -- South Africa -- LimpopoThesisNdou DL. Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools. []. , 2024 [cited yyyy month dd]. Available from:Ndou, D. L. (2024). <i>Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools</i>. (). . Retrieved fromNdou, Dakalo Lorraine. <i>"Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools."</i> ., , 2024.TY - Thesis AU - Ndou, Dakalo Lorraine AB - Moringa oleifera is a tree that has been studied extensively and it has been found to host a variety of medicinal and nutritional properties. Owing to these properties, it has often been referred to as a ‘miracle tree’. M. oleifera contains a variety of metabolites such as flavonoids, glucosinolates, phenolic acids, tannins, and carotenoids that are responsible for the pharmacological properties of this plant. In this study, the metabolite of interest in M. oleifera was the rutin flavonoid. Rutin has various pharmacological properties and its presence in M. oleifera makes the plant more bioavailable. In this study, the presence of rutin was evaluated in M. oleifera plants in households from different villages within the Vhembe District. The Molecular Networking (MN) approach was utilized to revisit the chemical space of flavonoids in M. oleifera. The aim was to establish the biochemical modifications responsible for the chemical diversity of these compounds, which have been reported to be associated with the purported pharmacological properties of this plant. Modern extraction methods such as ultrasonic-assisted aqueous two-phase extraction (UA-ATPE) and pipette tip micro-solid phase extraction (PT-μSPE) were explored in the extraction of the most bio-available and most sought-after flavonoid, rutin, in M. oleifera leaf extracts. This work has been divided into four experimental chapters. In Experimental Chapter 4, the presence of rutin was evaluated in the leaves of 135 M. oleifera plants from households in different villages within the Vhembe District of Limpopo Province of South Africa. The metabolite extraction was carried out using the conventional liquid extraction method using 80% MeOH and the resulting extracts were analyzed using UHPLC-qTOF-MS. The results from the UHPLC-qTOF-MS showed that only 15 plants produced rutin. This was confirmed using tandem mass spectrometry (MS2) and an authentic standard, which further validated the detected ion as a true representation of rutin. It was concluded that different cultivars of this plant are being grown in various households within the Vhembe District. These differences are expected to result in a negative perception towards Moringa plants, and as such, knowledge of the cultivar-chemical relationship should be made public with the intention to encourage the cultivation of proper plant species. The extraction of rutin from M. oleifera leaves using UA-ATPE was reviewed in Experimental Chapter 5. An ethanol/salt ATPE was formed for the extraction of rutin. Ultrasonication was used to assist in the extraction of rutin from the leaves. Central composite design (CCD) was used to design experiments and two factors which were optimized are ultrasonic time and ultrasonic temperature. The ammonium sulphate ((NH4)2SO4), sodium chloride (NaCl), and magnesium sulphate (MgSO4) salts were used to form the ethanol/salt ATPE two phase system. The resulting Response Surface Model (RSM) was observed to be a linear fit for the ethanol/(NH4)2SO4 and the ethanol/MgSO4 ATPE systems, with R2 values of 0.7339 and 0.5782, respectively, as obtained from the analysis of variance (ANOVA). The ethanol/NaCl ATPE system yielded a quadratic fit with R2 = 0.7865 and was observed to be the best performing in the extraction of rutin from the M. oleifera leaves with optimum extraction at a temperature of 25 ºC and time of 22.5 minutes. Based on multiple reaction monitoring (MRM) through the UHPLC-qTOF-MS technique, the concentration of rutin extracted by the ethanol/NaCl ATPE system was 240 μg L-1. According to ANOVA, temperature (the B-term) was found to be the significant term with a p < 0.0500 that the extraction of rutin through UA-ATPE is temperature-dependent. Therefore, it was concluded that the extraction of rutin from M. oleifera leaves favors low temperatures. The results of the current study further demonstrate the usefulness of simple extraction techniques, such as heated water with additives like salts, as a feasible method to enrich pharmacologically relevant metabolites from plants. This reaffirms traditional protocols that are currently used by communities which include boiling plants in water to extract useful chemical compounds for the treatment of common ailments such as colds and headaches. In Experimental Chapter 6, PT-μSPE was applied in the extraction of rutin from M. oleifera leaves using activated hollow carbon nanospheres (HCNSs) as the sorbent. The activated HCNSs were characterized using FTIR, which confirmed the presence of the functional groups of interest such as OH stretch, -COO- vibration, and C=O stretch. TGA thermogram showed a difference in the thermal stability of the raw and activated HCNSs, proving that the raw HCNSs are more stable than the activated HCNSs, and SEM displayed the difference in the morphology of the raw and activated HCNSs by observing the difference in the coalescence of the material. Parameters such as loading cycles, elution solvent, concentration of rutin, pH, loading volume, and mass of sorbent were optimized. The analysis of the extracts was conducted with a UV-Vis spectrophotometer to ascertain the recovery of rutin. The optimal conditions for rutin recovery using PT-μSPE were determined to be 15 loading cycles, i-PrOH as the elution solvent, a 2 ppm as standard concentration of rutin, a pH of 2, 500 μL as loading volume, and 1.5 mg of sorbent. The LOD, LOQ, and RSD values were found to be 0.604 mg L-1, 1.830 mg L-1, and 3.26%; respectively. It was thus confirmed that the PT-μSPE method is effective in the extraction of rutin even at trace levels based on the low LOD value obtained and the RSD value obtained proved that this method is a reliable pre-concentration technique and is thus repeatable for the analysis of complex samples. Therefore, in cases where some plants produce these compounds in minute concentrations, methods such as this one presented herein can be used to estimate and concentrate the pharmacologically relevant compounds. Molecular networking (MN) was used to study the global metabolic profile of M. oleifera and is outlined in Experimental Chapter 7. Herein, Global Natural Products Social (GNPS) platform was used to generate the MN from the LC-MS data obtained from the methanolic leaf extracts of M. oleifera. The MN was viewed and analyzed using Cytoscape. Through MN, it was observed that M. oleifera contains a variety of metabolites. Other GNPS tools such as network annotation propagation (NAP), DEREPLICATOR, MS2LDA, and MolNetEhancer were further used to compliment the classical MN model. To this end, MS2LDA was used to annotate the flavonoids found within M. oleifera. Kaempferol, quercetin, and isorhamnetin flavonoids were successfully annotated by MS2LDA. Additionally, Chrysin-6,8-C-diglucoside was also annotated and reported for the first time in M. oleifera leaves. The results of this study further suggest MN models as useful tools for chemical exploration, enabling the discovery of new metabolites by leveraging existing knowledge as "chemical charms" to unearth hidden metabolites. DA - 2024-09-06 DB - ResearchSpace DP - Univen LK - https://univendspace.univen.ac.za PY - 2024 T1 - Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools TI - Innovative Extraction and Identification of Rutin Flavonoid from Moringa oleifera Leaves Using UHPLC-qTOF-MS and Computational Metabolomics Tools UR - ER -