Purification of Chinese Sweet Leaf (R. suavissimus) Extracts through Alcohol Precipitation

The extract of Rubus suavissimus leaves contains bioactive compounds associated with antiangiogenic activity [1]. Because it was prepared by boiling water, it often contains a higher amount of polysaccharides, which contributes to impurity. In an effort to purify and concentrate this extract for elevated bioactivity, alcohol precipitation was used as a rapid and cost-effective purification method to remove polysaccharides. In this study, the sweet leaf tea crude extract powder was re-constituted into aqueous solutions, which were subjected to either filtration or no filtration pre-treatment prior to alcohol precipitation. Appropriate volumes of ethanol were added to the water extracts to yield 50%, 60%, 70%, 80%, 90%, and 95% ethanol precipitation. The supernatants (purified) and precipitants (impure) of each treatment were separated and dried to obtain dry weight. For the purified samples, quantitative analyses were performed for rubusoside, gallic acid, and ellagic acid contents using HPLC-PDA methods. As for precipitants, polysaccharides contents, expressed as glucose equivalent, were measured using the modified phenol-sulfuric acid colorimetric method [2]. A linear relationship between the precipitant yield and ethanol concentration was observed. As expected, as the alcohol concentrations increased from 50% to 95%, the weight of precipitants increased linearly approximately from 20% to 48%. The polysaccharides amount in the precipitants mirrored the weight changes. A clear difference in chemical compositions between the supernatant and precipitant was also observed from HPLC analysis. These results indicate that alcohol precipitation is a useful tool in the purification of plant extract such as the Chinese sweet leaf tea containing polysaccharides. Acknowledgement: The project described was partially supported by the McIntire-Stennis Cooperative Fund, the LSU Agricultural Center Technology Transfer Fund, and by Grant Number R21AT002882 from the National Center for Complementary & Alternative Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Complementary & Alternative Medicine or the National Institutes of Health. References: [1] Liu Z, et al. (2006) Phytother Res 20: 806–813. [2] Gao LJ, et al. (2004) Journal of Shandong Agricultural University (Natural Science) 35: 295–297.