Mota Joana, Figueira Maria E, Ferreira Ricardo B, Lima Ana
LEAF, Instituto Superior de Agronomia, Universidade de Lisboa, 1349-017 Lisbon, Portugal.
Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Foods. 2021 Jul 19;10(7):1663. doi: 10.3390/foods10071663.
One of the most challenging problems with food-borne bioactive compounds is that there are commonly no cost-effective, generally recognized as safe (GRAS) methods for obtaining gram quantities of their purified forms. Here we aimed at developing a method to isolate deflamin, an oligomeric protein from lupin seeds with anti-inflammatory and anticancer activity through matrix metalloprotease (MMP)-9 inhibition. Our goal was to develop a GRAS method that could be easily up-scalable whilst maintaining deflamin's activity. A sequential precipitation methodology was developed, using an aqueous extraction, followed by heat denaturation, acid precipitation and solubilization in ethanol. A final precipitation with 90% ethanol yielded a purified protein which was sequenced through mass spectrometry and tested for its MMP inhibitory activity using the Dye-quenched (DQ) gelatin assay and the standard wound healing assay in HT29 cells. The developed method yielded a purified oligomer, which represented 0.1% (/) of total dry seed weight and was positively confirmed to be deflamin. It further showed to effectively reduce MMP-9 gelatinolytic activity as well as colon cancer cell migration, hence corroborating the effectiveness of our method. Overall, this is the first reported method for isolating an MMP-9 inhibitor from legume seeds, which is up-scalable to an industrial level, in a cost-effective manner.
食源生物活性化合物面临的最具挑战性的问题之一是，通常没有经济高效、被普遍认为安全（GRAS）的方法来获得克级量的纯化形式。在此，我们旨在开发一种方法，从羽扇豆种子中分离出具有抗炎和抗癌活性的寡聚蛋白去火焰蛋白（deflamin），该活性通过抑制基质金属蛋白酶（MMP）-9来实现。我们的目标是开发一种GRAS方法，该方法易于扩大规模，同时保持去火焰蛋白的活性。我们开发了一种顺序沉淀方法，先进行水相提取，然后进行热变性、酸沉淀，最后在乙醇中溶解。用90%乙醇进行最终沉淀得到纯化蛋白，通过质谱对其进行测序，并使用染料淬灭（DQ）明胶分析和HT29细胞中的标准伤口愈合分析测试其对MMP的抑制活性。所开发的方法得到了一种纯化的寡聚物，其占种子总干重的0.1%（/），并被确认为去火焰蛋白。它进一步显示出能有效降低MMP-9的明胶分解活性以及结肠癌细胞的迁移，从而证实了我们方法的有效性。总体而言，这是首次报道的从豆类种子中分离MMP-9抑制剂的方法，该方法能够以经济有效的方式扩大到工业规模。
