Efficacy and mechanistic study of a carbohydrate derived fulvic acid with anti-inflammatory properties

Abstract

FAs (FA) are naturally occurring, refractory, complex organic acids that are reported to have several medicinal properties and are normally found in soils and water. They are available from health shops and pharmacies in several formulations with a number of claimed benefits but are not registered medicines. FAs are a product of the humification process of plant material originating in the environment from which these acids were obtained, but often include other organic compounds that are incorporated into the FA product. FAs have high carboxylic acid and hydroxyl content that enables stable complexation of different mineral ions that could lead to toxic heavy metal content, which can be harmful if consumed. This study assessed a synthetic FA, obtained using a patented controlled wet oxidation method of a metal-free food grade carbohydrate, to confirm biological activity and to profile the mass distribution of different components. Fulvimed SA (Pty) Ltd use good manufacturing practice and a patented process to synthesise large quantities of pure, high-quality FA in a liquid concentrate termed Carbohydrate-Derived FA (CHD-FA). Initial studies showed that this synthetic CHD-FA has more than 50 individual unidentified compounds, most of which appear to be carboxylic acids and common physiological metabolites, with no evidence of toxic compounds. This synthetic FA is aimed at the medicinal market as the FA that is obtained through this process has been shown to contain no toxic heavy metals. The main aim of this study was to determine whether a new more concentrated powdered formulation of this CHD-FA possesses similar anti-inflammatory properties to the previously tested less concentrated liquid formulation. The results from this study indicated that the new formulation of CHD-FA is effective in suppressing acute carrageenan-induced inflammation in the inflammatory rat footpad model. In addition, in vitro testing showed that CHD-FA exhibited a dose-dependent partial inhibition of complement receptor-3 (CR3) expression by activated neutrophils. Neutrophil adhesion to vascular endothelium or opsonised particles requires cell surface expression of CR3 receptors that is essential for neutrophil function. If cell adhesion is inhibited by the presence of CHD-FA, then extravascularisation of the neutrophils would be suppressed and extravascular inflammatory responses would be expected to be decreased. Further testing showed biologically relevant antioxidant properties using a lucigenin-enhanced chemiluminescence assay of stimulated neutrophils. Scavenging of the 2,2-azinobis (3-ethylbenzothiazoline 6-sulfonate) radical cation (ABTS۰+) using the TEAC assay confirmed the antioxidant activity of this new formulation of CHD-FA. Although not regarded as a modern technique, thin layer chromatography (TLC) was used to separate different FA batches to compare product reproducibility and in an attempt to obtain a profile of the compounds within the synthetic CHD-FA product. This was combined with a technique of direct elution from the TLC plates into a tandem -mass spectrometer to assess the mass distribution of the different spots obtained from the FAs. It has been demonstrated that CHD-FA possesses significant anti-inflammatory properties by in vitro and in vivo animal studies and confirmed in vitro antioxidant properties. These properties suggests that CHD-FA may be an effective immunomodulator for the treatment of diseases associated with an overproduction of reactive oxidants by human phagocytes such as the neutrophils. In addition, it can be seen that there are a large number of compounds in the FA with many co-eluting under different separating conditions and having a mass range extending beyond those previously reported. Despite the complex nature of CHD-FA, it has been shown to have several properties that indicate that it would act as an anti-inflammatory compound.