Preparation of Diterpenes‑Enriched Extract from Croton stellatopilosus Ohba Leaves using Enzyme‑ and Ultrasonic‑Assisted Extraction

Background: Croton stellatopilosus Ohba accumulates several diterpenes. Among them, plaunotol, a cytoprotective agent, has been prescribed to treat peptic ulcers. Objectives: We aimed to prepare the diterpene‑enriched extract (DEE) using enzyme‑ and ultrasonic‑assisted extractions. Materials and Methods: The type and amount of pectinase and cellulase and the condition of ultrasonic treatment were optimized. A combination of enzyme and ultrasonic treatments was performed. The high‑performance liquid chromatography (HPLC) method was validated and used for the determination of plaunotol and plaunolide contents in the DEE. Results: A combination of cellulase (10 units) and ultrasonic (at 80% amplitude) treatments enhanced the yield of plaunotol significantly. Application of the crude extract on diaion HP‑20 resin afforded the DEE. With the validated HPLC method, the DEE contained 8.81% ± 0.02% w/w of plaunotol and 8.40% ± 0.92% w/w of plaunolide. Conclusion: We established an efficient method to prepare DEE based on green extraction. This method showed about eightfold enhancement purification factor compared to the original crude extract and about 90% yield recovery.