Reflux extraction and column chromatography for rosmarinic acid- rich fraction from Orthosiphon stamineus

Background: Orthosiphon stamineus is a medicinal herb rich in caffeic acid derivatives. Rosmarinic acid is one of the caffeic acid-based phytochemicals which is also well-known for its antioxidant and anti-inflammatory properties. Objective: This study was focused on the preparation of rosmarinic acid-rich fraction to enhance the antioxidant capacity of O. stamineus. Method: The rosmarinic acid-rich fraction was prepared by using an integrated technique of reflux extraction and column chromatography. The concentration of ethanol was varied from 0-100 %v/v to optimize the yield of rosmarinic acid in the fraction. Results: This technique increased the rosmarinic acid content from 0.14 %w/w in the dried whole plant to 1.9 %w/w in the crude extract till 4.1 %w/w in the 80 %v/v ethanol fraction. The consecutive elution of packed column also produced eupatorin-rich fraction from 0.0008 %w/w in the dried whole plant to 0.17 %w/w in the crude extract and 0.5 %w/w in the 40 %v/v ethanol fraction. The increment of rosmarinic acid content enhanced the radical scavenging activity in the fractions. The IC50 was significantly decreased from 2424 to 37.7 ppm for the crude extract and its 80 %v/v ethanol fraction, respectively. Therefore, the increase of rosmarinic acid content also increased the antioxidant capacity of the plant extract. Conclusion: This rosmarinic acid-rich fraction could be further developed into various useful herbal products for the benefits of mankind