Fractionation of rosmarinic acid rich extract from orthosiphon stamineus for enzymatic anti-diabetic activity

Abstract

orthosiphon stamineus (o. stamineus) is one of the herbal plants containing high amount of rosmarinic acid. however, there is no standard guideline, specifically for rosmarinic acid rich extract from o. stamineus. therefore, this study was focused on the fractionation of rosmarinic acid from the o. stamineus crude extract using chromatographic methods, namely thin layer chromatography integrated with column chromatography (tlc-cc) and liquid-liquid extraction integrated with solid phase extraction (lle-spe). both fractionation methods were compared, and it was found that tlc-cc performed better than lle-spe to recover rosmarinic acid from the o. stamineus crude extract. the purity of the rosmarinic acid rich extract was 100% (fraction 8) which was greater than sub-fraction 1-3 (75%) prepared by lle-spe. tlc-cc increased the rosmarinic acid content from 3.6% w/w in crude extract to 100.0% w/w in the fraction 8, while lle-spe only increased the content of rosmarinic acid up to 75.0% w/w in the combined sub-fraction 1 to 3. the increase of rosmarinic acid content also simultaneously increased enzymatic anti-diabetic activities compared to the crude extract. the fraction of 100% rosmarinic acid from tlc-cc significantly (p < 0.05) inhibited the activity of a-amylase and a-glucosidase with ic50 values of 2.31 mg/ml and 0.34 mg/ml, respectively. the ic50 values were comparable to those results of standard drug, acarbose which showed 1.03 mg/ml and 1.66 mg/ml for a-amylase and a-glucosidase, respectively. kinetic studies revealed that the 100% rosmarinic acid fraction inhibited a-amylase competitively, but non-competitively for a-glucosidase inhibition. the introduction of rosmarinic acid as an inhibitor slow down the digestion rate of starch by a-amylases, and reduce the performance of a-glucosidase by decreasing its vmax value to 0.05 mm/min. understanding the kinetic information of the enzymatic reaction is important to predict in vivo metabolism of rosmarinic acid for drug design. in conclusion, high anti-diabetic property rosmarinic acid rich extract from o. stamineus can be obtained using column chromatography technology.