Ludexin, an edible anti-inflammatory and anticancer protein isolated from legume seeds

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Lupin protein concentrate as a novel functional food additive that can reduce colitis-induced inflammation and oxidative stress

Publication . Mota, Joana; Casimiro, Sandra; Fernandes, João; Hartmann, Renata M.; Schemitt, Elizângela; Picada, Jaqueline; Costa, Luis; Marroni, Norma; Raymundo, Anabela; Lima, Ana; Ferreira, Ricardo Boavida

Food fortification with bioactive compounds may constitute a way to ameliorate inflammatory bowel diseases (IBDs). Lupin seeds contain an oligomer named deflamin that can reduce IBD's symptoms via MMP-9 inhibition. Here, our goal was to develop a lupin protein concentrate (LPC) enriched in deflamin and to test its application as a food additive to be used as a functional food against colitis. The nutritional profile of the LPC was evaluated, and its efficacy in vivo was tested, either alone or as added to wheat cookies. The LPC presented high protein and carbohydrate contents (20.09 g/100 g and 62.05/100 g, respectively), as well as antioxidant activity (FRAP: 351.19 mg AAE/10 mg and DPPH: 273.9 mg AAE/10 mg). It was also effective against TNBS-induced colitis in a dose dependent-manner, reducing DAI scores by more than 50% and concomitantly inhibiting MMP-9 activity. When added to cookies, the LPC activities were maintained after baking, and a 4-day diet with LPC cookies induced a significant protective effect against acetic acid-induced colitis, overall bringing lesions, oxidative stress and DNA damage levels to values significantly similar to controls (p < 0.001). The results show that the LPC is an efficient way to deliver deflamin in IBD-targeted diets.

2022Journal article

Open access

Deflamin, an edible anti-inflammatory and anticancer protein isolated from legume seeds

Publication . Guerreiro, Joana Patrícia Mota; Ferreira, Ricardo Manuel de Seixas Boavida; Raymundo, Anabela Moreira; Lima, Ana Isabel Gusmão

Previous reports revealed a novel MMP-2 and -9 inhibitor in Lupinus albus seeds, named deflamin, with a strong potential as a nutraceutical for inflammatory bowel diseases (IBDs). In the present work our goal was 1) to better understand deflamin’s mode of action and 2) to produce an effective delivery system for it to be incorporated in IBD functional diets. Deflamin was found to be an oligomer comprising similar fragments from δ-conglutin and β-conglutin. It inhibited MMP-2 and -9 activities but not their gene expression and presented no cytotoxic effects. In vivo, it effectively reduced gelatinases activity in situ, hence minimizing the risk of secondary effects. Based on a previous patented method we were able to develop a lupin protein concentrate (LPC) which was used as an additive to cookies (10g/100g dough). Sweet and savory, as well as different flours (wheat, rice, buckwheat, oat, kamut and spelt) were tested. Overall, the LPC was found to improve the organoleptic and physical properties of the cookies. The biochemical compositions of all cookies with LPC presented higher protein and a golden-brown coloring. Furthermore, the aw and moisture content values suggested a high stability food product. Whilst the baking itself did not interfere with the gelatinase inhibitory activity in the cookies, the type of flour and the presence of sugar did, suggesting that the presence of carbohydrates can interfere with deflamin. Our final goal was tested the efficacy of the LPC in vivo, either alone or as added to cookies using two different types of colitis. Results confirmed that the LPC alone was effective against TNBS-induced colitis, in a dose dependent-manner, reducing more than 50% the DAI scores and inhibiting MMP-9 activity. When added to cookies, the LPC induced a significant protective effect against acetic acid-induced colitis, reducing lesion, oxidative stress and DNA damage levels. Overall, deflamin was shown to be an efficient and safe way to reduce IBDs symptoms, without prompting secondary effects. The developed LPC was found to be effective as a delivery system for deflamin and besides its activity towards MMP-9, the LPC further added a higher nutritional and antioxidant value to the already potential health benefits of deflamin. These findings can open new perspectives and novel approaches to tackle IBDS via functional diets.

2023Doctoral thesis

Open access

Lupinus albus protein componentes inhibit MMP-2 and MMP-9 gelatinolytic activity in vitro and in vivo

Publication . Mota, Joana; Direito, Rosa; Rocha, João; Fernandes, João; Sepodes, Bruno; Figueira, Maria Eduardo; Raymundo, Anabela; Lima, Ana; Ferreira, Ricardo Boavida

Matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) are regarded as important clinical targets due to their nodal-point role in inflammatory and oncological diseases. Here, we aimed at isolating and characterizing am MMP-2 and-9 inhibitor (MMPI) from Lupinus albus and at assessing its efficacy in vitro and in vivo. The protein was isolated using chromatographic and 2-D electrophoretic procedures and sequenced by using MALDI-TOF TOF and MS/MS analysis. In vitro MMP-2 and 9 inhibitions were determined on colon adenocarcinoma (HT29) cells, as well as by measuring the expression levels of genes related to these enzymes. Inhibitory activities were also confirmed in vivo using a model of experimental TNBS-induced colitis in mice, with oral administrations of 15 mg kg􀀀1. After chromatographic and electrophoretic isolation, the L. albus MMP-9 inhibitor was found to comprise a large fragment from -conglutin and, to a lower extent, small fragments of -conglutin. In vitro studies showed that the MMPI successfully inhibited MMP-9 activity in a dose-dependent manner in colon cancer cells, with an IC50 of 10 g mL􀀀1 without impairing gene expression nor cell growth. In vivo studies showed that the MMPI maintained its bioactivities when administered orally and significantly reduced colitis symptoms, along with a very significant inhibition of MMP-2 and -9 activities. Overall, results reveal a novel type of MMPI in lupine that is edible, proteinaceous in nature and soluble in water, and effective in vivo, suggesting a high potential application as a nutraceutical or a functional food in pathologies related to abnormally high MMP-9 activity in the digestive system

2021Journal article

Open access

Therapeutic Potential of Deflamin against Colorectal Cancer Development and Progression

Publication . Silva, Sara; Cavaco, Ana; Basso, Bianca; Mota, Joana; Cruz-Duarte, Raquel; Costa, Miguel; Carvalho, Lara; Lima, Ana; Costa, Luís; Ferreira, Ricardo; Martins, Marta

Matrix metalloproteinases (MMPs) are proteolytic enzymes that play a crucial role in tumor microenvironment remodeling, contributing to inflammatory and angiogenic processes, and ultimately promoting tumor maintenance and progression. Several studies on bioactive polypeptides isolated from legumes have shown anti-migratory, anti-MMPs, and anti-tumor effects, potentially constituting novel strategies for both the prevention and progression of cancer. In this work, we inves- tigated the anti-tumor role of deflamin, a protein oligomer isolated from white lupine seeds (Lupinus albus) reported to inhibit MMP-9 and cell migration in colorectal cancer (CRC) cell lines. We found that deflamin exerts an inhibitory effect on tumor growth and metastasis formation, contributing to increased tumor apoptosis in the xenotransplanted zebrafish larvae model. Furthermore, deflamin resulted not only in a significant reduction in MMP-2 and MMP-9 activity but also in impaired cancer cell migration and invasion in vitro. Using the xenograft zebrafish model, we observed that deflamin inhibits collagen degradation and angiogenesis in the tumor microenvironment in vivo. Overall, our work reveals the potential of deflamin as an agent against CRC development and progression.

2022Journal article

Open access