SIMULTANEOUS DETERMINATION OF ROSMARINIC ACID, SALVIANOLIC ACID B AND CAFFEIC ACID IN SALVIA LERIIFOLIA BENTH. ROOT, LEAF AND CALLUS EXTRACTS USING A HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY DETECTION TECHNIQUE

Abstract

Salvia leriifolia Benth. (Lamiaceae) is a medicinal plant with analgesic, hypoglycemic and anti-inflammatory properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. The present study aimed to determine the amounts of rosmarinic acid, salvianolic acid B and caffeic acid in the roots, leaves and calli of S. leriifolia using a simple, rapid and efficient HPLC method. In addition, the effect of different combinations of 2,4-dichlorophenoxyacetic acid and kinetin–as growth regulators–on phenolic composition was evaluated. The results indicated that the 3 phenolic acids were well separated using the applied gradient HPLC system [C18 (5mm) column; phosphoric acid 1% and methanol as mobile phase]. Limit of detection and quantification values for rosmarinic acid, caffeic acid and salvianolic acid B were 0.2 and 0.5, 1.0 and 2.0, and 0.5 and 1.0mg/mL, respectively. Inter- and intra-day variations for rosmarinic acid, caffeic acid and salvianolic acid B were 2.8% and 3.4%, 2.4% and 3.5%, and 4.8% and 4.7%, respectively. Recovery values for the 3 phenolic acids ranged from 96.3% to 98.9%. Rosmarinic acid was the most frequent phenolic acid in the ethanolic extract of roots, leaves and calli of S. leriifolia (0.33, 4.65 and 6.39 mg/g dry extract, respectively) followed by salvianolic acid B (0.11, 0.13 and 0.10 mg/g) and caffeic acid (0.15, 0.20 and 0.07 mg/g). The highest dry weight (0.226 g) of callus was achieved in medium containing 2 mg/L 2,4-D and 1 mg/L KIN. To conclude, the present study indicated that rosmarinic acid is the major phenolic acid in the roots, leaves and calli of S. leriifolia, using a simple, rapid, efficient and reproducible HPLC method for the separation and simultaneous determination of phenolic acids. Moreover, the content of evaluated phenolics was found to be higher in the leaf extract vs. root and callus extracts of the plant.