Dataset Information

Biochemical and Phylogenetic Analysis of Italian Phaseolus vulgaris Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

ABSTRACT: Phaseolus vulgaris α-amylase inhibitor (α-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian α-amylase activity, reducing the absorption of dietary carbohydrates. Numerous studies have reported the efficacy of preparations based on this protein on the control of glycaemic peaks in type-2 diabetes patients and in overweight subjects. A positive influence on microbiota regulation has also been described. In this work, ten insufficiently studied Italian P. vulgaris cultivars were screened for α-amylase- and α-glucosidase-inhibiting activity, as well as for the absence of antinutritional compounds, such as phytohemagglutinin (PHA). All the cultivars presented α-glucosidase-inhibitor activity, while α-AI was missing in two of them. Only the Nieddone cultivar (ACC177) had no haemagglutination activity. In addition, the partial nucleotide sequence of the α-AI gene was identified with the degenerate hybrid oligonucleotide primer (CODEHOP) strategy to identify genetic variability, possibly linked to functional α-AI differences, expression of the α-AI gene, and phylogenetic relationships. Molecular studies showed that α-AI was expressed in all the cultivars, and a close similarity between the Pisu Grogu and Fasolu cultivars' α-AI and α-AI-4 isoform emerged from the comparison of the partially reconstructed primary structures. Moreover, mechanistic models revealed the interaction network that connects α-AI with the α-amylase enzyme characterized by two interaction hotspots (Asp38 and Tyr186), providing some insights for the analysis of the α-AI primary structure from the different cultivars, particularly regarding the structure-activity relationship. This study can broaden the knowledge about this class of proteins, fuelling the valorisation of Italian agronomic biodiversity through the development of commercial preparations from legume cultivars.

SUBMITTER: Peddio S

PROVIDER: S-EPMC10457751 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Biochemical and Phylogenetic Analysis of Italian <i>Phaseolus vulgaris</i> Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

Peddio Stefania S Lorrai Sonia S Padiglia Alessandra A Cannea Faustina B FB Dettori Tinuccia T Cristiglio Viviana V Genovese Luigi L Zucca Paolo P Rescigno Antonio A

Plants (Basel, Switzerland) 20230811 16

<i>Phaseolus vulgaris</i> α-amylase inhibitor (α-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian α-amylase activity, reducing the absorption of dietary carbohydrates. Numerous studies have reported the efficacy of preparations based on this protein on the control of glycaemic peaks in type-2 diabetes patients and in overweight subjects. A positive influence on microbiota regulation has also been described. In this work, ten insufficiently studied Italian < ...[more]

PMID: 37631130

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Project description:BackgroundPhaseolamin or α-amylase inhibitor 1 (αAI) is a glycoprotein from common beans (Phaseolus vulgaris L.) that inhibits some insect and mammalian α-amylases. Several clinical studies support the beneficial use of bean αAI for control of diabetes and obesity. Commercial extracts of P. vulgaris are available but their efficacy is still under question, mainly because some of these extracts contain antinutritional impurities naturally present in bean seeds and also exhibit a lower specific activity αAI. The production of recombinant αAI allows to overcome these disadvantages and provides a platform for the large-scale production of pure and functional αAI protein for biotechnological and pharmaceutical applications.ResultsA synthetic gene encoding αAI from the common bean (Phaseolus vulgaris cv. Pinto) was codon-optimised for expression in yeasts (αAI-OPT) and cloned into the protein expression vectors pKLAC2 and pYES2. The yeasts Kluyveromyces lactis GG799 (and protease deficient derivatives such as YCT390) and Saccharomyces cerevisiae YPH499 were transformed with the optimised genes and transformants were screened for expression by antibody dot blot. Recombinant colonies of K. lactis YCT390 that expressed and secreted functional αAI into the culture supernatants were selected for further analyses. Recombinant αAI from K. lactis YCT390 was purified using anion-exchange and affinity resins leading to the recovery of a functional inhibitor. The identity of the purified αAI was confirmed by mass spectrometry. Recombinant clones of S. cerevisiae YPH499 expressed functional αAI intracellularly, but did not secrete the protein.ConclusionsThis is the first report describing the heterologous expression of the α-amylase inhibitor 1 (αAI) from P. vulgaris in yeasts. We demonstrated that recombinant strains of K. lactis and S. cerevisiae expressed and processed the αAI precursor into mature and active protein and also showed that K. lactis secretes functional αAI.

Project description:Ipomea aquatica, also known as water spinach, is an aquatic non-conventional leafy vegetable and is considered a healthy and seasonal delicacy in ethnic food culture. The study revealed the presence of rich chemical and biochemical composition in I. aquatica and antioxidant activities. Moreover, the plant extracts demonstrated significant DNA damage prevention activity against UV/H2O2-induced oxidative damage. High-resolution mass spectrometric analysis by UPLC-qTOF-MS/MS resulted in the identification of over 65 different compounds and 36 important secondary metabolites. Most of the compounds identified represented polyphenolic compounds, viz. polyphenol glycosides and phenolic acids, followed by alkaloids and terpenoids. A UPLC-DAD method was developed and quantified for 10 different polyphenolic compounds. Out of all the metabolites examined, a significant number of compounds were reported to have various bioactive properties, including antibacterial, antiviral, antitumor, hepatoprotection, and anti-depressant effects. The plant extracts were found to contain various compounds, including euphornin, lucidenic acid, and myricitin glycosides, which possess significant medicinal value. Metabolite analysis utilizing GC-MS revealed the presence of various fatty acids, amino acids, sugars, and organic acids. The analysis revealed the presence of essential unsaturated fatty acids such as α-linolenic acid as well as beneficial substances such as squalene., The evaluation of glycemic control activity was carried out by comprehending the inhibitory potential of α-amylase and α-glucosidase, outlining the kinetics of the inhibition process. The inhibitory activities were compared to those of acarbose and revealed stronger inhibition of α-glucosidase as compared to α-amylase. Furthermore, the mechanism of inhibition was determined using in silico analysis, which involved molecular docking and molecular dynamic simulation of the identified IA phytochemicals complexed with the hydrolase enzymes. The study generates convincing evidence that dietary intake of I. aquatica provides a positive influence on glycemic control along with various health-protective and health-promoting benefits.

Project description:Diabetes mellitus is a chronic disease that is becoming a serious global health problem. Diabetes has been considered to be one of the major risks of cataract and retinopathy. Synthetic and natural product inhibitors of carbohydrate degrading enzymes are able to reduce type 2 diabetes and its complications. For a long time, potatoes have been portrayed as unhealthy for diabetic patients by some nutritionist due to their high starch content. However, purple and red potato cultivars have received considerable attention from consumers because they have high levels of polyphenolic compounds that have potent antioxidant activities. In this study, we screened the total phenolics (TP) and total anthocyanins (TA) and analyzed the phenolic and anthocyanin compounds in selected potato cultivars and advanced selections with distinct flesh colors (purple, red, yellow and white). Purple and red potato cultivars had higher levels of TP and TA than tubers with other flesh colors. Chlorogenic acid is the predominant phenolic acid, and major anthocyanin is composed of the derivatives of petunidin, peonidin, malvidin and pelargonidin. We tested the potential inhibitory effect of potato extracts on the activities of α-amylase and α-glucosidase, which were targeted to develop antidiabetic therapeutic agents. We also measured inhibitory effect of potato extracts on aldose reductase (AR) which is a key enzyme that has been a major drug target for the development of therapies to treat diabetic complications. Purple flesh tubers extract showed the most effective inhibition of α-amylase, α-glucosidase, and aldose reductase with IC50 values 25, 42, and 32 μg/ml, respectively. Kinetic studies showed that anthocyanins are noncompetitive inhibitors of these enzymes, whereas phenolic acids behaved as mixed inhibitors for α-amylase and α-glucosidase and noncompetitive inhibitors for AR. This study supports the development of a positive and healthful image of potatoes, which is an important issue for consumers.

Dataset Information

Biochemical and Phylogenetic Analysis of Italian Phaseolus vulgaris Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

Publications

Biochemical and Phylogenetic Analysis of Italian <i>Phaseolus vulgaris</i> Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

Similar Datasets

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