Synthesis, characterisation and properties of fulvic acid, derived from a carbohydrate

PhD (Pharmacy with Pharmaceutical Chemistry), North-West University, Potchefstroom Campus Introduction: Human and animal health is constantly threatened by pathogenic microbes and the emergence of bacteria resistant to standard medicinal interventions has escalated the search for new and innovative pharmaceutical solutions. Natural medicines have received much attention in recent years as a potential answer to the problem of “super bugs”. Fulvic acid is a composition of organic acids found in nature and known for its anti-inflammatory, antimicrobial and antioxidant properties. Unfortunately, the detection of heavy metals embedded in the molecular structure of fulvic acids extracted from numerous environmental sources has rendered it unsafe for medicinal applications. A new invention by Fulhold Pharma Ltd to synthetically produce fulvic acid from sucrose, identified as Carbohydrate-Derived Fulvic Acid (CHD-FA), is a major international breakthrough in the production of a heavy metal free fulvic acid. CHD-FA is produced through a non-catalytic wet oxidation process and complies with standardised product specifications for molecular consistency and safety. CHD-FA has anti-inflammatory, antimicrobial and antioxidant therapeutic health benefits. Purpose: * To propose a theoretical model for the compound identified as the major constituent of CHD-FA. * The identification of the backbone structures embedded in CHD-FA. * To review the pharmacological properties of CHD-FA based on the composition of the backbone structures embedded in the molecular composition of CHD-FA’s cluster structure with the emphasis on anti-inflammatory, antibacterial, antifungal, antiviral and antioxidant properties. Methods: * A theoretical model, based on literature, was developed to describe the mechanisms involved in the non-catalytic wet oxidation process that has transformed sucrose into the major component (anhydrofulvic acid) of CHD-FA. Carbohydrate-Derived Fulvic Acid * Samples were collected for each of the different phases of the non-catalytic wet oxidation process, from the start-up of the reactor to the start of the exothermic reaction. Nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-tandem mass spectrometry (LC-MSMS) was used to analyse these samples to identify molecular changes during the various stages of this process. * Gas chromatography-mass spectrometry (GC-MS), Fourier’s transform infrared (FTIR) and NMR were used to provide general information on the mixture. * Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used to desorb and ionise CHD-FA without fragmentation, in order to identify and measure the absolute molecular weight of the main component in CHD-FA. * LC-MSMS was used to identify the most prominent backbone structures embedded in the CHD-FA molecular structure. These compounds were mainly identified by injecting a sample of CHD-FA in the LC-MSMS, identifying the major mass ions, generating empirical formulas associated with these peaks and then using software to predict molecular structures associated with these compounds. * The clinical applications associated with the anti-inflammatory, antimicrobial, antifungal, antiviral and antioxidant properties of CHD-FA were assessed through a comprehensive literature review of the characteristics of the backbone structures in the molecular structure of CHD-FA. Results: Objective 1: The detailed description of the theoretical pathway for the synthesis of the major component of CHD-FA, namely molecular fulvic acid, has provided evidence that the non-catalytic wet oxidation synthetic process of sucrose to produce molecular fulvic acid is a one-pot synthesis process consisting of a myriad of chemical reactions in the reactor. Colour changes in the reactor solution have confirmed the theoretical pathway description of a step-by-step process. The colours changed progressively from a light yellow to a dark brown colour. NMR and LC-MSMS analyses have confirmed that the colour changes demonstrated the transformation of sucrose into molecular fulvic acid. GCMS analysis revealed a concord between the structure of CHD-FA and penicillin-derivated fulvic acid. The MALDI-TOF MS identified 308 g/mol as the highest intensity peak with a natural abundance of 20.8 % in the spectrum and confirmed it as the most prominent component in CHD-FA. Batch-to-batch consistency of CHD-FA was recorded by chromatographic and spectroscopic data for more than 30 production runs over a four year period. Carbohydrate-Derived Fulvic Acid Objective 2: Similarities between the spectroscopic data of CHD-FA and literature data from environmental fulvic acids were indicated by FTIR and 13C NMR. However, CHD-FA has unique characteristics which differentiate it from environmental fulvic acids. CHD-FA has more carboxyl, ester, amide and aliphatic carbons in its molecular structure compared to the fulvic acid reference standards from the International Humic Substances Society. GC-MS confirmed the complexity of the molecular CHD-FA structure. The chromatogram overlay of CHD-FA and the reference standard, penicillin-derived fulvic acid (CAS 479-66-3), confirmed the presence of fulvic acid in CHD-FA. The most prominent component of the molecular structure of CHD-FA shown by LC-MSMS spectrum is 7,8‐dihydroxy‐3‐methyl‐10‐oxo‐1H,10H‐pyrano[4,3‐b]chromene‐9‐carboxylic acid with the empirical formulae of C14H10O7. This component is the dehydrated analogue of fulvic acid (C14H12O8) indicative of a loss of a water molecule during sample preparation. The LC-MSMS shows molecular ion m/z 290 and 24 prominent peaks, which represents the key structures in CHD-FA. It is evident that CHD-FA is a cluster of organic compounds. 24 prominent peaks were characterised as the backbone structures embedded in CHD-FA. This, with reference to the molecular composition of CHD-FA, is the most significant finding of the present study. Malic acid, maleic acid, levulinic acid, succinic acid, propenoic acid, phthalic acid, arabonic acid, itaconic acid, glucuronic acid, glutaric acid, benzene tri- and tetracarboxylic acids were identified as the backbone structures of CHD-FA. These backbone structures are interlinked with each other and with the parent structure via intermolecular bonding to form a cluster molecular structure. Objective 3: A comprehensive literature review of the clinical properties of the backbone structures of CHD-FA has demonstrated anti-inflammatory, antibacterial, antifungal, antiviral and antioxidant properties. These properties are therefore embedded in CHD-FA. Conclusion: CHD-FA, derived synthetically from sucrose through a non-catalytic wet oxidation process, is a pure form of fulvic acid. CHD-FA has the same medicinal properties as penicillin-derived reference standard fulvic acid and fulvic acids derived from various environmental sources. The identification of the twenty four backbone structures embedded in the supramolecular structure of CHD-FA is evidence that CHD-FA is a cluster of organic structures. This cluster of organic structures is responsible for the unique characteristics of CHD-FA, which include anti- Carbohydrate-Derived Fulvic Acid inflammatory, antimicrobial and antioxidant properties. The batch-to-batch consistency demonstrated for the manufacturing of CHD-FA in this study offers much potential for the use of CHD-FA as a natural pharmaceutical compound in the development of natural medicines. Doctoral