Cryodos freeze dryer

The ripe fruits of V. vinifera (Bangalore Blue, Muscat variety) were collected from Tirupathi, Andhra Pradesh, India, during October 2012 and authenticated by Dr. K. Madhava Chetty, Botanist, Sri Venkateswara University, Tirupati, India. The seeds were deposited in Herbarium of Department of Botany, Sri Venkateswara University with the number 86783. The seeds were separated from pulp and shade dried. The dried powdered seed materials (2 kg) were subjected to maceration in 5 L of ethanol (95%) for 3 days at room temperature and filtered into a clean round bottom flask using an adsorbent cotton wool and a filter paper (Whatman no. A-1). The whole process was repeated five times to ensure maximum yield of ethanol soluble compounds from the seed powder. The combined ethanolic extract was concentrated at 40–50°C using a rotary evaporator (Rotavapor R-210, Buchi, Switzerland) and lyophilized using a cryodos freeze dryer (Telstar, Barcelona, Spain) to yield 26.67 g of solid extract (1.28% W/W). These steps ensure complete evaporation of ethanol, leaving behind a solid mass [15 (link)].

In order to obtain an extract including total polyphenols, the extraction was performed using the acid MeOH, according to the method published by Genskowsky et al. [22 (link)], with slight modifications. The initial sample consisted of 50 g lyophilized powdered wild maqui fruit (seed and pulp) from a packaged and commercialized product (Isla Natura de Chile^®, Chiloé, Chile). In total, 250 mL of MeOH/H⁺ (0.1% HCl) at pH 1 were added to the sample, and then homogenized with an ultrasound device (Hielscher Ultrasound Technology UP400S, Teltow, Germany). The extract was centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was collected. This procedure was repeated 5 times. The supernatants were mixed in a round-bottomed flask and evaporated until dryness using a rotary evaporator (Büchi B-490, Hampton, VA, USA). The dried extract was dissolved in distilled water and centrifuged at 4000 rpm for 4 min. Finally, it was passed through 2 filtering processes (100–150 MM and 40–100 MM) and then lyophilized in a Telstar Cryodos Freeze Dryer (Tokyo, Japan). The final extract was stored at −20 °C. All experiments were carried out under darkness and controlled temperature.