Analysis of sterols and sterolins in hypoxis hemerocallidea and related herbal medicine

Abstract

Phytosterols and their glucosides (sterolins) have many therapeutic indications e.g. immune modulation, hypercholesterolaemia and benign prostatic hyperplasia (BPH). In this study sterolslsterolins in three BPH phytotherapeutics (Hypoxis hemerocallidea, Prunus africana and Serenoa repens) and related products were investigated. The aim of this study was to develop, evaluate and apply TLC and HPLC methods for the qualitative and quantitative analyses of sterols and sterolins. A new optimum TLC method was developed for good visibility and separation of phytosterols and sterolins and could be used to qualitatively compare sterol/sterolin content. A published HPLC method to determine the bioavailability of β-sitosterol in humans was used in a new application to quantitatively determine phytosterols in plant extracts. A new and sensitive method to determine hypoxoside (norlignan diglucoside unique to Hypoxidaceae), by isolation from the crude methanol extract with solid phase extraction (SPE) and HPLC quantification using fluorescence detection (excitation wavelength of 230 nm and emission wavelength of 345 nm), was developed. The developed TLC and adapted HPLC methods were applied to determine the stability of phytosterols, subjected to increased temperature and gamma irradiation. Phytosterols in isolated form were more stable than the phytosterols in plant material. The data from the accelerated stability tests could be used to estimate the shelf-lives of the BPH phytotherapeutics and related sterol containing products. The HPLC method to determine β-sitosterol in serum, was evaluated during a pilot study of a clinical trial, to test the bio-equivalence of different phytosterol containing products. The method was found not sensitive enough to determine β-sitosterol in serum, notwithstanding improvements made, Le. changing the extraction ratio; experimenting with higher dosages, and different products. As result, the proposed clinical trial could not be performed, in the future, serum could rather be analysed by gas chromatographic methods. TLC and HPLC analyses of medicinal African potato tea, indicated that it contained hypoxoside, but not β-sitosterol or β-sitosterolin. β-Sitosterol (accepted to be the active of H. hemerocallidea) might not be the main active in African potato tea. Hypoxoside and a compound (red spot compound), noticed on TLC plates of acetone extracts of Prunus africana, Serenoa repens, Moducare®, Harzol®, Immunochoice® and Nutricare®, were extracted with water. This general presence of the red spot compound could point to a possible important function. Preparative TLC was unsuccessful to isolate the red spot compound, but column chromagraphy was successfully applied. From the proton and carbon NMR spectra, it was concluded, that the compound was definitely not a steroid and could either be a coumarin or an isoflavanoid, with a sugar unit (possibly a rhamose) attached to it. Further analyses to elucidate the structure failed due to decomposition of the compound. Further work on structure elucidation is required and possible therapeutic activity should also be investigated. The sterols and sterolins in H. hemerocallidea and related herbal medicine can be qualitatively and quantitatively analysed with the developed TLC and adapted HPLC methods. This provides natural medicine industry with necessary procedures to ensure proper quality, safety and stability.