Optimized Moringa Seed Extract Preparation

Moringa seeds were obtained from the Jamaica Moringa Farmers’ Association (Kingston, Jamaica). A voucher specimen was prepared and submitted to the Rutgers University CHRB Chrysler Herbarium (Accession number: 146375). The preparation of MSE was optimized based on the degree of biotransformation of GLSs to ITCs in situ, in order to obtain the highest MIC-1 content. The optimization was performed prior to preparation of the extract for in vivo and in vitro studies. The development and optimization of the procedure for the preparation of MSE involved incubating ground seeds in water at a controlled temperature with constant agitation for a duration of time, after which ethanol was added at 4x the volume of water to arrest the myrosinase reaction (Table 1). Incubation temperatures were first evaluated at 25°C or 37°C using a solvent ratio of 1:4 (g seeds:mL H₂O) and incubation time of 2 h. Then solvent ratio was evaluated at 1:2, 1:3, and 1:4 using an incubation temperature of 37°C and incubation time of 2 h. Incubation time was also evaluated at 0.5, 1 and 2 h using an incubation temperature of 37°C and a solvent ratio of 1:4. All experiments were performed in triplicates using 10 g of ground seeds. Seed/solvent slurries were then filtered through a Whatman filter paper, and filtrate was reduced in volume via rotary evaporation and dried via lyophilization. The freeze-dried extracts were weighed and analyzed for MIC-1 content by LC-MS (see below).
A large batch of MSE, obtained using optimized extraction conditions (1 g seed powder:3 mL water at 37°C for 2 h with constant agitation, followed by addition of 4x volume of ethanol, filtered and dried), was produced and stored at -20°C and used for all subsequent studies. The extract was analyzed and MIC-1 quantified by LC-MS. Nutritional analysis and fatty acid composition of MSE was performed by NJ Feed Labs (Trenton, NJ).