Optimization and validation of folate extraction from Moringa oleifera leaves powder

Moringa oleifera leaves are potentially benign sources of natural folates, thus a reliable folate extraction protocol is necessary, which directly affects the accuracy of subsequent determination of total folates or various folate derivatives. With this aim, process variables including antioxidants, extraction buffer pH, Moringa oleifera leaves powder (MOLP)/extraction buffer ratio, heating extraction time, amounts of α-amylase, protease and rat serum, and also incubation time of rat serum were investigated and optimized by response surface methodology (RSM). It is interesting that no positive influence of α-amylase or protease solution was observed in this study, consequently a single-enzyme treatment was used. An average folate level of 1190.06 µg/100 g dry matter was achieved by microbial assay under the optimized conditions of extraction buffer pH 7.0, MOLP/extraction buffer ratio 1:24 (g/mL), rat serum 350 µL, digest time 2.0 h, in good agreement with the value predicted by quadratic model. In addition, the validity of protocol established in this study was confirmed. The intra-day precision (relative standard deviation) varied between 1.42% with 1.84%, inter-day precision varied in the range of 1.34–2.25%. The average recoveries were in the range of 101.56–103.27%. Besides, the matrix effects ranged from 101.41 to 103.34%. The protocol could provide a reference to fairly evaluate folates in Moringa oleifera leaves from diverse sources, and even in other leafy plants. Meanwhile, Moringa oleifera leaves with high folate level seem to have tremendous potential to increase nutritional value of foods as well as to enhance regional economies.