Application of Hibiscus sabdariffa extract to manage obesity

Global rise in obesity has necessitated continued effort to develop suitable therapies for its effective management. Although a number of synthetic medications with anti-obesity properties are commercially available, they are associated with wide range of risks and side effects. Many plant-derived compounds with potential preventive and therapeutic effects against obesity are identified with negligible side effects. Phenolic compounds from plant sources have shown to possess promising anti-obesity effects assessed through cell line, animal, and clinical trials. In addition, hydroxycitric acid, commonly extracted from Garcinia cambogia is marketed as weight managing supplement. Considering the presence of high concentration of phenolic compounds and hydroxycitric acid, extract of Hibiscus sabdariffa can be considered as suitable material for quantifying anti-obesity effects. Therefore, this PhD study aimed to optimize the extraction of phenolic compounds and hydroxycitric acid from H. sabdariffa followed by the determination of their anti-obesity potential individually and in combination. For this purpose, in vitro studies i.e., inhibition of digestive enzymes and cell line studies on inhibition of adipogenesis were undertaken.  The first experimental chapter focused on the extraction of bioactive compounds (phenolic compounds and hydroxycitric acid) and determining their physicochemical characteristics. The type and concentration of solvents and extraction temperature varied to maximise the yield and antioxidant potency of phenolic compounds. Spectrophotometric and chromatographic methods were used for determining phenolic and flavonoid contents, and antioxidant activity. Low temperature (23°C) and organic solvents (methanol and ethanol) were found to be suitable medium for extracting phenolic and flavonoid compounds with higher antioxidant activity. The antioxidant activity of these phenolic compounds was found to be stable for 5 weeks at -30oC. Hydroxycitric acid was extracted in the form of potassium salt to overcome its instability. The purity of extracted potassium hydroxycitrate was 180.14 μg/mg of dry extract. The second experimental chapter focused on determining the inhibition of digestive enzymes by the phenolic compounds and potassium hydroxycitrate individually and in combination. For this, three enzymes associated with obesity (α-amylase, α-glucosidase and pancreatic lipase) were selected and the inhibition potential of the extract was determined through in vitro assays. All phenolic extracts extracted using aqueous and organic (methanol, ethanol, and ethyl acetate) solvents showed different degree of inhibition against these enzymes. The ethyl acetate, methanolic and ethanolic extracts showed higher inhibition efficacy (IC50) for α-amylase (3.69 mg/mL), α-glucosidase (1.59 mg/mL) and pancreatic lipase (1.88 mg/mL) respectively as compared to aqueous extract (5.74, 3.65 and 4.25 mg/mL) which could be attributed to the higher concentration of phenolic compounds in the organic extracts. These phenolic compounds had binding affinity with these enzymes at their active sites which reduced their activity. Potassium hydroxycitrate did not show inhibition against these enzymes when used alone or in combination with phenolic compounds. The third experimental chapter assessed the inhibition of adipogenesis/adipocyte differentiation by extracted phenolic compounds and potassium hydroxycitrate in human adipose-derived stem cells (hADSCs). Adipogenesis was chemically induced in hADSCs for 21 days and the cells were treated with different concentration of phenolic extracts and potassium hydroxycitrate during first three days of differentiation. The lipid accumulation after completion of adipogenesis was quantified. Phenolic extracts showed significant reduction (45-95%) in the lipid accumulation in these cells at the concentration of 1 mg/mL, however, potassium hydroxycitrate had no inhibiting effect on adipogenesis in hADSCs. The adipogenic inhibition by phenolic compounds was reflected on the down regulation of gene expression of two key adipogenic markers [peroxisome proliferator-activated receptor- γ (PPAR-γ) and aP2].  The incorporation or presence of hydroxycitrate in phenolic extract did not show additional inhibition of adipogenesis in hADSCs. The fourth experimental chapter focussed on developing suitable encapsulating shell material for these phenolic compound-rich extracts of H. sabdariffa in order to extend their shelf life and also to make them available in easy-to-handle bead form. Encapsulation of phenolic extract was achieved through ionic gelation using alginate-chitosan hybrid matrix. The concentration of alginate, chitosan and extract was optimized to obtain the highest encapsulation efficiency. The maximum encapsulation efficiency (91.9%) of phenolic compounds was achieved with 3% alginate, 0.2% chitosan and 1% extract concentrations. The capsule beads had a smooth surface with no cracks and pores on the surface. The alginate-chitosan matrix created through ionic gelation preserved antioxidant activity of phenolic compounds (81%) as compared to the non-encapsulated extracts (71%). This matrix also contributed to the sustained release of phenolic compounds by delivering more than 50% of the compounds to the simulated intestinal condition. These findings from this research provide scientific basis for application of phenolic compound rich extracts of H. sabdariffa as natural therapeutic agent to manage obesity.