Acanthus mollis rhizomes were collected in their plant vegetative stage in September 2017 in La Isla, Colunga (Asturias, Spain) (43°48′39.5″ N–5°22′48.13″ W). All sampled plants were over 10 years old. Plant material was identified and authenticated by the Botanical Department of San Pablo–CEU University in Madrid. A voucher specimen (Ref. 3372) was deposited into the Faculty of Pharmacy Herbarium, San Pablo–CEU University in Madrid. The major development of leaves and rhizomes, together with the long photoperiod and high intensity of light, made September an appropriate period for the collection of rhizomes [42 (link)]. The rhizomes were dried at room temperature (20–25 °C) and then spread evenly and turned over every 24 h. When the humidity was reduced to values lower than 10–12%, the rhizomes were ground into powder using a Culatti grinder equipped with a 1 mm link filter (Janke and Kunkel GMGH, Staufen, Germany). The ground material was extracted three times with hexane in an ultrasonic bath (Ultrasons, Selecta®, Barcelona, Spain) at 50 °C for 1 h in a solvent-to-solid ratio of 10mL/g. Hexane was chosen as a solvent due to its attributes, such as a simple recovery, non-polar nature, and low latent heat of vaporisation. This enabled the extraction of non-polar molecules of great pharmacological interest [43 (link)]. The extract was filtered through Whatman no. 1 filter paper and subsequently evaporated to dryness under a reduced pressure at <50 °C using a Buchi rotary evaporator. The concentrated extract was then dried to a constant weight under a stream of cold air at room temperature (22 °C) and stored at 4 °C until use. The extract yield was 0.37% (w/w).
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