Isolation of a bioactive compound for acute respiratory distress syndrome, sakuranetin, from Baccharis retusa through High Speed Countercurrent Chromatography

According to Ministry of Health, lung disorders affect more than 6.4 million people in Brazil [1]. With delicate clinical management, for critical episodes of respiratory failure, bronchodilators and anti-inflammatory drugs (corticosteroids) are introduced, which, although they interrupt a patient's clinical emergency, bring numerous medium and long-term harms [2]. Among the studies aiming to find alternatives to the treatment of lung disorders, a plant of Genus Baccharis, found in the Brazilian Atlantic Forest, has shown promising results. Among the compounds present in B. retusa, the flavanone sakuranetina stands out, which, in addition to it activity against pulmonary disorders [3], was attributed to also display antiparasitic, antifungal, antiviral, anti-inflammatory, antioxidant, and others activities. Aiming the optimization of isolation of sakuranetin from B. retusa, in this project we intend to evaluate the use of countercurrent chromatography (CCC). Previously, several biphasic CCC solvent systems were evaluated [4], among which the system containing equal parts hexane, ethyl acetate, methanol and water (HEMWat) was the most suitable, according to ITO’s golden rules [5]. Thus, the dichloromethane phase of the B. retusa leaf ethanol extract (BRED - 500 mg) was subjected to fractionation through High-Speed Countercurrent Chromatography (HSCCC-HTPrep - Quattro), at reverse phase mode, in which the stationary phase is the organic phase, and operating at a flow rate of 2 mL.min-1 at 860 rpm. The percentage of stationary phase retention, under these operating conditions, ranged from 75 to 83%, suggesting a satisfactory separation resolution [5]. During elution and extrusion, one column volume (Vc - 95 mL) was collected for each process, resulting in 50 total fractions, which were pooled into 12 groups (BRED 1 – 12) according to their similarity by TLC. It was observed that the compound of interest was isolated at groups BRED 10 and 11 (108 mg), 98% pure (HPLC) and the identity of the compound was confirmed by TLC, HPLC and NMR. Thus, in conclusion the countercurrent chromatography technique has allowed the high purity and rapid isolation of this compound, in a single hundred-minute step, allowing future prospects for large-scale isolation and development of formulations for lung disorders, specifically asthma and pulmonary emphysema.

References

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4-de CAMPOS, M.L.O.S.. et al. Optimization in the isolation of sakuranetin and other phenolics from Baccharis retusa (Asteraceae) by Countercurrent Chromatography: SS selection and K value determination. CCC2018, Braunschweig - Germany, August 1-3, 2018.
5-ITO, Y. Golden rules and pitfalls in selecting optimum conditions for high-speed Counter-current chromatography. Journal of Chromatography A. 1065, 2, 145 to 168 – 2005.