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1.
Food Res Int ; 163: 112155, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36596106

RESUMO

Inhibiting carbohydrate-hydrolyzing enzymes has been considered as an effective approach for controlling starch digestion and postprandial blood glucose level. α-Amylase and amyloglucosidase (AMG) are commonly applied in analysis of starch digestion behaviour. Catechins have been shown with the inhibiting effects on α-amylase. However, the inhibitory activity of catechins against AMG needs to be further studied. Therefore, AMG inhibition of 8 catechins and the mechanisms were studied in this work through substrate depletion, inhibition kinetics, molecular docking, fluorescence quenching, differential scanning calorimetry, and isothermal titration calorimetry. The inhibitory activity of catechins with galloyl moiety (CGMs) was found to be lower than the corresponding catechins without the moiety (Cs). All catechins were anti-competitive inhibitors, indicating that they tended to bind with AMG-starch complex in the digestion system, rather than with AMG directly. Interestingly, CGMs had higher quenching effects on AMG fluorescence than Cs, due to the additional π-stacking between aromatic rings of GM and AMG fluorophores. Also, CGMs had a higher binding affinity to AMG, due to the tendency of GM to AMG active site, although the affinity was much weaker than that of starch to AMG. Besides, catechins did not affect AMG thermostability. Therefore, there was an inconsistency between catechins-AMG binding interactions and the enzyme inhibition because the predominant sites for catechins binding were the non-active sites on AMG-starch complex, rather than the enzyme active ones. Conclusively, inhibition mode should also be considered when evaluating the inhibitory activity of a polyphenol based on the polyphenol-enzyme binding affinity.


Assuntos
Catequina , Polifenóis , Polifenóis/farmacologia , Catequina/química , Glucana 1,4-alfa-Glucosidase , Simulação de Acoplamento Molecular , alfa-Amilases/metabolismo , Amido/química
2.
J Dairy Sci ; 106(2): 1002-1012, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36543642

RESUMO

The objective of this study was to determine the effects of including exogenous amylolytic or fibrolytic enzymes in a diet for high-producing dairy cows on in vitro ruminal fermentation. Eight dual-flow continuous-culture fermentors were used in a replicated 4 × 4 Latin square. The treatments were control (CON), a xylanase and glucanase mixture (T1), an α-amylase mixture (T2), or a xylanase, glucanase, and α-amylase mixture (T3). Treatments were included at a rate of 0.008% of diet dry matter (DM) for T1 and T2 and at 0.02% for T3. All treatments replaced the equivalent amount of soybean meal in the diet compared with CON. All diets were balanced to have the same nutrient composition [30.2% neutral detergent fiber (NDF), 16.1% crude protein (CP), and 30% starch; DM basis], and fermentors were fed 106 g/d divided into 2 feedings. At each feeding, T2 was pipetted into the respective fermentor and an equivalent amount of deionized water was added to each fermentor to eliminate potential variation. Experimental periods were 10 d (7 d for adaptation and 3 d for sample collection). Composite samples of daily effluent were collected and analyzed for volatile fatty acids (VFA), NH3-N, and lactate concentrations, degradability of DM, organic matter, NDF, CP, and starch, and flow and metabolism of N. Samples of fermentor contents were collected from each fermentor at 0, 1, 2, 4, 6, and 8 h after feeding to determine kinetics of pH, NH3-N, lactate, and VFA concentrations over time. All data were analyzed using PROC GLIMMIX of SAS (SAS Institute Inc.), and the repeated variable of time was included for kinetics measurements. Treatment did not affect mean pH, degradability, N flow and metabolism, or the concentrations of VFA, NH3-N, or lactate in the effluent samples. Treatment did not affect pH, acetate:propionate ratio, or the concentrations of lactate, NH3-N, total VFA, acetate, propionate, butyrate, isobutyrate, valerate, or caproate. However, the concentration of total VFA tended to change at each time point depending upon the treatment, and T2 tended to have a greater proportion of 2-methylbutyrate and isovalerate than CON, T1, or T3. As 2-methylbutyrate and isovalerate are branched-chain VFA that are synthesized from branched-chain amino acids, T2 may have an increased fermentation of branched-chain amino acids or decreased uptake by fibrolytic microorganisms. Although we did not observe changes in N metabolism due to the enzymes, there could be changes in microbial populations that utilize branched-chain VFA. Overall, the tested enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows.


Assuntos
Lactação , Propionatos , Animais , Bovinos , Feminino , alfa-Amilases/metabolismo , Ração Animal/análise , Dieta/veterinária , Digestão , Ácidos Graxos Voláteis/metabolismo , Fermentação , Lactatos/metabolismo , Propionatos/metabolismo , Rúmen/metabolismo , Amido/metabolismo
3.
Arch Oral Biol ; 145: 105574, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36395562

RESUMO

OBJECTIVES: Salivary statherin and alpha-amylase play significant roles in biofilm formation and pathogenic bacteria adhesion. Examination of these proteins may provide information on their roles in periodontal diseases. The present study was based on the hypothesis that; the salivary proteins -statherin and alpha-amylase- effective on biofilm formation, may play important roles in the etiology of periodontal disease. Therefore, we aimed to analyze the differences in periodontal diseases compared to periodontal health in order to search their roles in periodontal disease. METHODS: Patients with gingivitis (n = 26) and periodontitis (n = 20), and periodontally healthy individuals (n = 21) were included in this study. Unstimulated whole saliva samples were obtained from a total of 67 individuals. Salivary statherin level and alpha-amylase activity were determined using ELISA and enzymatic methods, respectively. RESULTS: Statherin levels in saliva were significantly higher in the periodontitis group compared to the gingivitis group (p = 0.014), while alpha-amylase activities and total protein levels were slightly higher in the periodontitis and gingivitis groups compared to controls, without significant differences among the groups (p = 0.295 and p = 0.019, respectively). Statherin levels showed positive correlations with gingival and plaque indices in the disease groups. CONCLUSIONS: The results suggest that statherin level in saliva increase to provide a protective effect against periodontitis, and higher salivary statherin level is related to the degree of gingival inflammation and plaque accumulation.


Assuntos
Gengivite , Doenças Periodontais , Periodontite , Humanos , alfa-Amilases/metabolismo , Aderência Bacteriana , Gengivite/metabolismo , Doenças Periodontais/metabolismo , Periodontite/metabolismo , Saliva/metabolismo
4.
Chem Biol Interact ; 370: 110312, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36535312

RESUMO

In this study, eight new compounds (7a-h) based on triazole compounds containing ester groups were synthesized with high yields. The structures of the synthesized compounds (7a-h) were elucidated by various spectroscopic methods (element analysis, FT-IR, 1H-(13C) NMR). Antioxidant, anticancer, and α-amylase enzyme inhibition activities of synthesized new triazole derivatives were carried out, and the effects of different groups on the activity were investigated. When the determined antioxidant properties of the compounds were examined, all synthesized compounds showed a moderate radical scavenging effect against radicals depending on the concentration (6.25-200 g/mL). All compounds except the three derivatives were found to have higher IC50 values than the standard drug acarbose (IC50: 891 µg/mL) according to the α-amylase enzyme inhibition results. Compound 7g (IC50: 50 g/mL) was discovered to have nearly eighteen (18) times the activity of the conventional medication acarbose (IC50: 891 µg/mL). Compounds synthesized for anticancer activity studies were screened against the Hela cell line, and the results were compared with standard cis-platinum (IC50: 16.30 µg/mL). Compound 7g (IC50: 19.78 µg/mL) was found to have almost the same activity as cis-platinum. Using Qikprop, the compounds were thoroughly tested for ADME qualities, and none violated any drug similarity standards. According to ADME data, whole physicochemical drug-likeness parameters of molecules remained within defined ranges as stipulated in the Lipinski rules (RO5) and revealed a high bioavailability profile. The molecular docking results with 2QV4 and 4GQR alpha-amylase enzymes demonstrated that all molecules have a high affinity, indicating polar and apolar interaction with critical amino acids in the α-amylase binding pocket.


Assuntos
Acarbose , Antioxidantes , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Antioxidantes/farmacologia , Células HeLa , Cisplatino , Triazóis/farmacologia , Triazóis/química , Espectroscopia de Infravermelho com Transformada de Fourier , alfa-Amilases/metabolismo , Estrutura Molecular
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 289: 122251, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36542921

RESUMO

Developing naturally active components to control α-amylase/α-glucosidase activity is highly desired for preventing and managing type 2 diabetes. Rapeseed oil is rich in active phenolic compounds and seed oil is a major source of liposoluble inhibitors to these enzymes. However, it remains unclear about the interaction of phenolic compounds in rapeseed oil with α-amylase/α-glucosidase. This study found that the important phenolic compounds from rapeseed oil (Sinapic acid, SA; canolol, CAO; canolol dimer, CAO dimer) possessed effective inhibition performance against α-amylase and α-glucosidase. CAO showed the lowest and highest inhibitory effect, respectively. In the kinetics studies, the inhibition mechanism of SA/CAO/CAO dimer against α-glucosidase was non-competitive, exhibiting a different way from α-amylase. Fluorescence quenching spectra implied that the static processes were responsible for the spontaneous binding between the compounds and enzymes. Fourier-transform infrared spectroscopy (FT-IR) displayed these compounds-induced conformation alterations of α-amylase/α-glucosidase. Molecular docking revealed that SA/CAO/CAO dimer decreased the catalytic efficiency of α-amylase/α-glucosidase through hydrogen bonds, hydrophobic force, or π-π interaction. Molecular dynamics matched well with the experimental and docking results regarding the inhibitory behaviors and interactions toward α-amylase/α-glucosidase. These results demonstrated the potential benefits of phenolic compounds from rapeseed oil in antidiabetic-related activities.


Assuntos
Diabetes Mellitus Tipo 2 , Simulação de Dinâmica Molecular , Humanos , Simulação de Acoplamento Molecular , Inibidores de Glicosídeo Hidrolases/química , alfa-Glucosidases/metabolismo , Óleo de Brassica napus , alfa-Amilases/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
6.
ACS Synth Biol ; 12(1): 196-202, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36580286

RESUMO

Developing effective bacterial autolytic systems for fast release of intracellular bioproducts could simplify purification procedures and help with the high throughput screening of mutant libraries in protein engineering. Here, we developed a fast and tightly regulated E. coli autolytic system, named the FhuD-lysozyme-SsrA mediated autolytic (FLSA) system, by integrating the secretion signal peptide, T7 lysozyme, and E. coli ClpX/P-SsrA protein degradation machinery. To decrease the cytotoxicity of leaky T7 lysozymes, the SsrA tag was fused to the C-terminus of T7 lysozyme to confer a tight regulation of its production. Using sfGFP as a reporter, we demonstrated that anchoring the Sec-Tat dual pathway signal peptide FhuD to the N-terminus of T7 lysozyme-SsrA could give the highest cell lysing efficiency. The optimization of the FLSA system indicated that weak alkaline conditions (pH 8.0) and 0.5% Triton X-100 could further increase the lysing efficiency by about 24%. The FLSA system was validated by efficient production of sfGFP and human growth hormone 1 (hGH1) in a shake flask, with a cell lytic efficiency of approximately 82% and 80%, respectively. Besides, the FLSA system was applied for large-scale fermentation, in which approximately 90% sGFP was released with a cell density OD600 of 110. Moreover, the FLSA system was also tested for α-amylase mutant library screening in microplates, and the results showed that intracellular α-amylase can be efficiently released out of cells for activity quantitation. In all, the FLSA system can facilitate the release of intracellular recombinant proteins into the cell culture medium, which has the potential to serve as an integrated system for large-scale production of recombinant targets and high throughput enzyme engineering in synthetic biology.


Assuntos
Escherichia coli , Muramidase , Humanos , Escherichia coli/metabolismo , Muramidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Sinais Direcionadores de Proteínas , alfa-Amilases/metabolismo
7.
Molecules ; 27(24)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36557873

RESUMO

In the CAZy database, the α-amylase family GH13 has already been divided into 45 subfamilies, with additional subfamilies still emerging. The presented in silico study was undertaken in an effort to propose a novel GH13 subfamily represented by the experimentally characterized cyclomaltodxtrinase from Flavobacterium sp. No. 92. Although most cyclomaltodextrinases have been classified in the subfamily GH13_20. This one has not been assigned any GH13 subfamily as yet. It possesses a non-specified immunoglobulin-like domain at its N-terminus mimicking a starch-binding domain (SBD) and the segment MPDLN in its fifth conserved sequence region (CSR) typical, however, for the subfamily GH13_36. The searches through sequence databases resulted in collecting a group of 108 homologs forming a convincing cluster in the evolutionary tree, well separated from all remaining GH13 subfamilies. The members of the newly proposed subfamily share a few exclusive sequence features, such as the "aromatic" end of the CSR-II consisting of two well-conserved tyrosines with either glycine, serine, or proline in the middle or a glutamic acid succeeding the catalytic proton donor in the CSR-III. Concerning the domain N of the representative cyclomaltodextrinase, docking trials with α-, ß- and γ-cyclodextrins have indicated it may represent a new type of SBD. This new GH13 subfamily has been assigned the number GH13_46.


Assuntos
Flavobacterium , alfa-Amilases , alfa-Amilases/metabolismo , Flavobacterium/genética , Flavobacterium/metabolismo , Sequência de Aminoácidos , Glicosídeo Hidrolases/metabolismo , Filogenia
8.
Molecules ; 27(21)2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36364077

RESUMO

To combat emerging antimicrobial-resistant microbes, there is an urgent need to develop new antimicrobials with better therapeutic profiles. For this, a series of 13 new spiropyrrolidine derivatives were designed, synthesized, characterized and evaluated for their in vitro antimicrobial, antioxidant and antidiabetic potential. Antimicrobial results revealed that the designed compounds displayed good activity against clinical isolated strains, with 5d being the most potent (MIC 3.95 mM against Staphylococcus aureus ATCC 25923) compared to tetracycline (MIC 576.01 mM). The antioxidant activity was assessed by trapping DPPH, ABTS and FRAP assays. The results suggest remarkable antioxidant potential of all synthesized compounds, particularly 5c, exhibiting the strongest activity with IC50 of 3.26 ± 0.32 mM (DPPH), 7.03 ± 0.07 mM (ABTS) and 3.69 ± 0.72 mM (FRAP). Tested for their α-amylase inhibitory effect, the examined analogues display a variable degree of α-amylase activity with IC50 ranging between 0.55 ± 0.38 mM and 2.19 ± 0.23 mM compared to acarbose (IC50 1.19 ± 0.02 mM), with the most active compounds being 5d, followed by 5c and 5j, affording IC50 of 0.55 ± 0.38 mM, 0.92 ± 0.10 mM, and 0.95 ± 0.14 mM, respectively. Preliminary structure-activity relationships revealed the importance of such substituents in enhancing the activity. Furthermore, the ADME screening test was applied to optimize the physicochemical properties and determine their drug-like characteristics. Binding interactions and stability between ligands and active residues of the investigated enzymes were confirmed through molecular docking and dynamic simulation study. These findings provided guidance for further developing leading new spiropyrrolidine scaffolds with improved dual antimicrobial and antidiabetic activities.


Assuntos
Anti-Infecciosos , Antioxidantes , Antioxidantes/química , Simulação de Acoplamento Molecular , Quinoxalinas , Hipoglicemiantes/farmacologia , Hipoglicemiantes/química , Antibacterianos/química , Anti-Infecciosos/farmacologia , Relação Estrutura-Atividade , alfa-Amilases/metabolismo
9.
Nutrients ; 14(21)2022 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-36364940

RESUMO

Bioactive peptides may exert beneficial activities in living organisms such as the regulation of glucose metabolism through the inhibition of alpha amylases. Algae and cyanobacteria are gaining a growing interest for their health-promoting properties, and possible effects on glucose metabolism have been described, although the underlying mechanisms need clarification. This study proposes a computer-driven workflow for a proteome-wide mining of alpha amylase inhibitory peptides from the proteome of Chlorella vulgaris, Auxenochlorella protothecoides and Aphanizomenon flos-aquae. Overall, this work presents an innovative and versatile approach to support the identification of bioactive peptides in annotated proteomes. The study: (i) highlighted the presence of alpha amylase inhibitory peptides within the proteomes under investigation (including ELS, which is among the most potent inhibitory tripeptides identified so far); (ii) mechanistically investigated the possible mechanisms of action; and (iii) prioritized further dedicated investigation on the proteome of C. vulgaris and A. flos-aquae, and on CSSL and PGG sequences.


Assuntos
Chlorella vulgaris , Cianobactérias , Proteoma , alfa-Amilases Pancreáticas , Peptídeos/farmacologia , Peptídeos/metabolismo , Cianobactérias/química , Cianobactérias/metabolismo , alfa-Amilases/metabolismo , Glucose
10.
Org Biomol Chem ; 20(45): 8962-8976, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36326164

RESUMO

Due to the growth in the incidence of diabetes mellitus throughout the world, the urgency in the search for new safe and effective inhibitors of α-amylase and α-glucosidase is increasing. In this work, we attempted to carry out studies on the synthesis, modification and comprehensive computer and biological research of new derivatives of monothiooxamides. It was shown that monothiooxamides based on 3-aminopyridin-2(1H)-ones enter into transamidation reactions with hydrazine hydrate to form the corresponding thiohydrazides. Furthermore, under the action of chloroacetyl chloride and succinic anhydride, these thiohydrazides are cyclized into conjugated 1,3,4-thiadiazole derivatives. The possible biological activity of the obtained products was evaluated by molecular docking using the AutoDock Vina program. Compounds 7a and 8b showed higher binding affinities for selected target proteins compared to the known anti-diabetic drugs acarbose and TAK-875. The obtained new derivatives of 1,3,4-thiadiazole showed sufficiently high values of inhibitory activity in the in vitro test against the enzymes α-amylase and α-glucosidase as well as sufficiently low IC50 values (for 8b 122.2 µM), which is 8 times less than the value for the reference drug acarbose (998.3 µM). All synthesized derivatives of monothiooxamides 5-8(a-c) showed no cytotoxic properties at physiological concentrations in the MTT test in human neonatal dermal fibroblasts. Moreover, some compounds (6a-c, 7a-c, 8b,c) showed pronounced cytoprotective activity. Thus, the compounds 5-8(a-c) synthesized by us, both according to the results of computer calculations and in vitro biological screening, showed their potential antidiabetic activity and high prospects for further in-depth studies, including in vivo studies.


Assuntos
Tiadiazóis , alfa-Glucosidases , Humanos , Acarbose , alfa-Amilases/química , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tiadiazóis/farmacologia , Tiadiazóis/química
11.
Int J Mol Sci ; 23(21)2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36362160

RESUMO

Six α-amylase/subtilisin inhibitor genes (MnASIs) were identified from mulberry (Morus notabilis). In this study, bioinformatics and expression pattern analysis of six MnASIs were performed to determine their roles in resistance to B. cinerea. The expression of all six MnASIs was significantly increased under Botrytis cinerea infection. MnASI1, which responded strongly to B. cinerea, was overexpressed in Arabidopsis and mulberry. The resistance of Arabidopsis and mulberry overexpressing MnASI1 gene to B. cinerea was significantly improved, the catalase (CAT) activity was increased, and the malondialdehyde (MDA) content was decreased after inoculation with B. cinerea. At the same time, H2O2 and O2- levels were reduced in MnASI1 transgenic Arabidopsis, reducing the damage of ROS accumulation to plants. In addition, MnASI1 transgenic Arabidopsis increased the expression of the salicylic acid (SA) pathway-related gene AtPR1. This study provides an important reference for further revealing the function of α-amylase/subtilisin inhibitors.


Assuntos
Arabidopsis , Morus , Arabidopsis/genética , Arabidopsis/metabolismo , Morus/genética , Morus/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Doenças das Plantas/genética , Botrytis/metabolismo , Subtilisinas/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo , Resistência à Doença/genética
12.
PLoS One ; 17(10): e0276984, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301972

RESUMO

BACKGROUND AND OBJECTIVE: Annona muricata L. peel has been recognized for many ethnobotanical uses, including diabetes management. However, limited detailed scientific information about its mechanism of antidiabetic activity exists. The objective of this study was to evaluate the anti-diabetic properties of an aqueous extract of A. muricata peel (AEAMP) and its mechanism of action on alloxan-induced diabetic rats. METHODS: In vitro antidiabetic assays, such as α-amylase and α-glucosidase were analyzed on AEAMP. Alloxan monohydrate (150 mg/kg b.w) was used to induce diabetes in the rats. 150 mg/kg b.w positive control group doses of 6.67, 13.53, and 27.06 mg/kg were administered to 3 groups for twenty-one days. The positive control group was administered 30 mg/kg of metformin. The negative and normal control groups were administered distilled water. The fasting blood glucose, serum insulin, lipid profile, inflammatory cytokines, antioxidant markers, carbohydrate metabolizing enzymes, and liver glycogen were analyzed as well as PI3K/AKT and apoptotic markers PCNA and Bcl2 by RT-PCR. RESULTS: AEAMP inhibited α-amylase and α-glucosidase enzymes more effectively than acarbose. AEAMP reduced FBG levels, HOMA-IR, G6P, F-1,6-BP, MDA, TG, TC, AI, CRI, IL-6, TNF-α, and NF-κB in diabetic rats. Furthermore, in diabetic rats, AEAMP improved serum insulin levels, HOMA-ß, hexokinase, CAT, GST, and HDL-c. Liver PI3K, liver PCNA and pancreas PCNA were not significantly different in untreated diabetic rats when compared to normal rats suggesting alloxan induction of diabetes did not downregulate the mRNA expression of these genes. AEAMP significantly up-regulated expression of AKT and Bcl2 in the liver and pancreatic tissue. It is interesting that luteolin and resorcinol were among the constituents of AEAMP. CONCLUSIONS: AEAMP can improve ß-cell dysfunction by upregulating liver AKT and pancreatic PI3K and AKT genes, inhibiting carbohydrate metabolizing enzymes and preventing apoptosis by upregulating liver and pancreatic Bcl2. However, the potential limitation of this study is the unavailability of equipment and techniques for collecting more data for the study.


Assuntos
Annona , Diabetes Mellitus Experimental , Hipoglicemiantes , Extratos Vegetais , Animais , Ratos , Aloxano/farmacologia , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo , Annona/química , Apoptose , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Hipoglicemiantes/uso terapêutico , Inflamação/tratamento farmacológico , Insulinas/sangue , Fosfatidilinositol 3-Quinases/metabolismo , Extratos Vegetais/uso terapêutico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima
13.
J Agric Food Chem ; 70(41): 13223-13232, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36206318

RESUMO

Bioaccessibility of plant sterols (PS) in an enriched wholemeal rye bread was evaluated, for the first time, using the INFOGEST protocol without gastric lipase (GL) and cholesterol esterase (CE), with GL or GL + CE. Moreover, human chewing and an in vitro oral phase (simulated salivary fluid and α-amylase) were evaluated for this purpose. The addition of GL decreased the bioaccessibility of total PS (from 23.8 to 18.5%), whereas the use of GL + CE does not significantly affect PS bioaccessibility. The in vitro oral phase resulted in an ineffective homogenization of the fresh vs partially dried and milled bread, reducing the bioaccessibility of total (from 20.2 to 12.8%) and individual PS. The INFOGEST digestion including the use of GL and CE, as well as an oral phase with human chewing, is proposed for the assessment of PS bioaccessibility in a solid matrix such as wholemeal rye bread since it more closely approximates the in vivo situation.


Assuntos
Pão , Fitosteróis , Humanos , Fitosteróis/metabolismo , Secale/metabolismo , Metabolismo dos Lipídeos , Esterol Esterase/metabolismo , Fase Oral , Triticum/metabolismo , alfa-Amilases/metabolismo , Lipase/metabolismo , Digestão
14.
J Agric Food Chem ; 70(42): 13709-13718, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36238980

RESUMO

Maltotetraose (G4) is composed of four glucose units linked by the α-1,4-glycosidic bond, which has excellent adaptability in food processing and specific physiological functions. Maltotetraose-forming amylases (MFAses) are used in the industry as a promising tool for G4 production. The MFAse from Pseudomonas saccharophila STB07 (MFAPS), which belongs to the GH13, can preferentially hydrolyze substrates to G4. MFAPS contains a carbohydrate-binding module (CBM). In this study, we removed the CBM to obtain the mutant MFAPS-ΔCBM. We explored the aspects affecting the catalytic performance of enzymes through structural simulations and molecular docking. Results showed that when the CBM was removed, the thermal stability of MFAPS was slightly reduced, and its catalytic ability for long-chain substrates, such as corn starch, was significantly reduced. However, the catalytic ability and product specificity of the substrates with shorter chain length, such as maltodextrin (DE 7-9), were improved. The G1-G7 (glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6), and maltoheptaose (G7)) contents and G4 proportion of the mutant MFAPS-ΔCBM reaction at 24 h were 11.1 and 11.6% higher than those of MFAPS, respectively. The results also showed that the forces of MFAPS on the substrate near the -4, -1, +1, and +3 subsites were critical for its product specificity.


Assuntos
Maltose , Pseudomonas , Pseudomonas/genética , Pseudomonas/metabolismo , Simulação de Acoplamento Molecular , Oligossacarídeos/química , Glucose , Amido , Amilases/metabolismo , Especificidade por Substrato , alfa-Amilases/metabolismo
15.
Front Endocrinol (Lausanne) ; 13: 1022623, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36313779

RESUMO

In an attempt to find new targets for α-amylase and α-glucosidase for the treatment of type 2 diabetes mellitus, the present study aims in determining the anti-diabetic potential of synthesized dihydropyrimidinone derivatives. The in vitro α-glucosidase and α-amylase inhibitory activity was performed and the molecular docking analysis of the ligand in the active binding site of target protein was determined. The results revealed significant percent inhibition of α-glucosidase by the compound 6-benzyl-4-(4-hydroxyphenyl)-3,4,6,7-tetrahydro-1H-pyrrolo[3,4-d]pyrimidine-2,5-dione (compound A). The active compound showed 81.99% inhibition when compared to standard ascorbic acid having percent inhibition 81.18%. The IC50 of active compound (A) showed to be 1.02 µg/ml. The molecular docking analysis revealed that the ligand bound to the active binding site of protein with the lowest binding energy of -7.9 kcal/mol that was also significantly similar to standard having -7.8 kcal/mol binding energy. The molecular dynamic simulation studies also revealed stable binding of ligand in the active binding site of protein with low RMSD of 1.7 Å similar to the protein RMSD 1.6Å In conclusion, the study revealed a potential new target against α-glucosidase to treat type 2 diabetes mellitus.


Assuntos
Diabetes Mellitus Tipo 2 , alfa-Glucosidases , Humanos , alfa-Glucosidases/metabolismo , Simulação de Acoplamento Molecular , Ligantes , Diabetes Mellitus Tipo 2/tratamento farmacológico , alfa-Amilases/metabolismo
16.
Future Med Chem ; 14(21): 1507-1526, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36268762

RESUMO

Background: To discover novel lead molecules against diabetes, Alzheimer's disease and oxidative stress, a library of arylated pyrazole-fused pyran derivatives, 1-20, were synthesized in a one-pot reaction. Materials & methods:1H-NMR spectroscopic and electron ionization mass spectrometry techniques were used to characterize the synthetic hybrid molecules 1-20. Analogs were screened against four indispensable therapeutic targets, including α-amylase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase enzymes. Results: Except for derivatives 17 and 18, all other compounds exhibited varying degrees of inhibitory activities against target enzymes. The kinetic studies revealed that the synthetic molecules followed a competitive-type mode of inhibition for α-amylase and acetylcholinesterase enzymes, as well as a non-competitive mode of inhibition for α-glucosidase and butyrylcholinesterase enzymes. In addition, molecular docking studies identified crucial binding interactions of ligands with the enzyme's active site. Conclusion: These molecules may serve as a potential drug candidate to cure diabetes, Alzheimer's disease and oxidative stress in the future.


Assuntos
Doença de Alzheimer , Diabetes Mellitus , Humanos , Butirilcolinesterase/metabolismo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Simulação de Acoplamento Molecular , Inibidores da Colinesterase/química , alfa-Glucosidases/metabolismo , Piranos/uso terapêutico , Cinética , alfa-Amilases/metabolismo , Pirazóis/uso terapêutico , Relação Estrutura-Atividade , Estrutura Molecular
17.
Sci Rep ; 12(1): 17847, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36284171

RESUMO

Ionizing radiation can not only reduce the yield of rice but also cause rice toxicity, and consumption of this kind of rice threatens human health. Moreover, the production and application of freon has further caused a hole in the earth's ozone layer, increasing the amount of ionizing radiation from the sun affecting rice. To select and breed new radiation-resistant rice varieties, dry seeds of the indica-japonica subspecies of tetraploid rice subjected to different doses of ionizing radiation were investigated for their responses during germination. The results showed that the relative water absorption, seed vigour and GA3 content sharply decreased in response to three different doses of ionizing radiation, and the regulation of the expression of genes related to α-amylase synthesis and gibberellin metabolism was disrupted. Moreover, the degree of inhibition increased with increasing dose. Notably, under 3.0 × 1017 ions/cm2 radiation, an upregulation of OsGA3ox2 expression resulted in a sharp increase in GA3 content in the indica-japonica tetraploid rice, and upregulated expression of OsAmy3A and OsAmy3D resulted in sharp increase in α-amylase activity, water absorption, and sucrose and fructose contents, which resulted in the seed vigour being greater than that of its parents. The results indicate that additional research on the physiological and molecular features of indica-japonica tetraploid rice seed germination in response to ionizing radiation is needed.


Assuntos
Oryza , Humanos , Oryza/metabolismo , Germinação/fisiologia , Sementes/metabolismo , Giberelinas/metabolismo , Tetraploidia , Ozônio Estratosférico , Melhoramento Vegetal , alfa-Amilases/genética , alfa-Amilases/metabolismo , Íons/metabolismo , Sacarose/metabolismo , Água/metabolismo , Frutose/metabolismo
18.
BMC Complement Med Ther ; 22(1): 235, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36064352

RESUMO

BACKGROUND: Lysiphyllum strychnifolium (Craib) A. Schmitz, a traditional Thai medicinal plant, is mainly composed of polyphenols and flavonoids and exhibits several pharmacological activities, including antioxidant, anticancer, antimicrobial, and antidiabetic activities. However, the mechanism by which pure compounds from L. strychnifolium inhibit glucose catalysis in the small intestine and their effect on the glucose transporter remain unknown. METHODS: The objectives of this research were to examine the effect of 3,5,7-trihydroxychromone-3-O-𝛼-L-rhamnopyranoside (compound 1) and 3,5,7,3',5'-pentahydroxy-flavanonol-3-O-𝛼-L-rhamnopyranoside (compound 2) on the inhibition of α-amylase and α-glucosidase, as well as glucose transporters, such as sodium-glucose cotransporter 1 (SGLT1), glucose transporter 2 (GLUT2), and glucose transporter 5 (GLUT5), using Caco-2 cells as a model of human intestinal epithelial cells. Additionally, the binding affinity and interaction patterns of compounds against two receptor proteins (SGLT1 and GLUT2) were determined for the first time utilizing a molecular docking approach. RESULTS: In the α-amylase inhibition assay, a concentration-dependent inhibitory response was observed against the enzyme. The results indicated that compound 1 inhibited α-amylase activity in a manner similar to that of acarbose (which exhibit IC50 values of 3.32 ± 0.30 µg/mL and 2.86 ± 0.10 µg/mL, respectively) in addition to a moderate inhibitory effect for compound 2 (IC50 = 10.15 ± 0.53 µg/mL). Interestingly, compounds 1 and 2 significantly inhibited α-glucosidase and exhibited better inhibition than that of acarbose, with IC50 values of 5.35 ± 1.66 µg/mL, 510.15 ± 1.46 µg/mL, and 736.93 ± 7.02 µg/mL, respectively. Additionally, α-glucosidase activity in the supernatant of the Caco-2 cell monolayer was observed. In comparison to acarbose, compounds 1 and 2 inhibited α-glucosidase activity more effectively in Caco-2 cells without cytotoxicity at a concentration of 62.5 µg/mL. Furthermore, the glucose uptake pathways mediated by SGLT1, GLUT2, and GLUT5- were downregulated in Caco-2 cells treated with compounds 1 and 2. Additionally, molecular modeling studies revealed that compounds 1 and 2 presented high binding activity with SGLT1 and GLUT2. CONCLUSION: In summary, our present study was the first to perform molecular docking with compounds present in L. strychnifolium extracts. Our findings indicated that compounds 1 and 2 reduced glucose uptake in Caco-2 cells by decreasing the expression of glucose transporter genes and inhibiting the binding sites of SGLT1 and GLUT2. Therefore, compounds 1 and 2 may be used as functional foods in dietary therapy for postprandial hyperglycemia modulation of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Fabaceae , Acarbose , Células CACO-2 , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucose/metabolismo , Humanos , Hipoglicemiantes/farmacologia , Simulação de Acoplamento Molecular , Polifenóis , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismo
19.
Biomed Pharmacother ; 153: 113510, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36076597

RESUMO

Balanites aeqyptiaca (BA) seeds were toasted at 70 °C, milled and the oil expelled to resolve to meal which were defatted to resolve to defatted balanites aeqyptiaca (BA) protein meal and (BA) protein concentrate respectively. These were subjected to analysis using standard methods. There exist marked trend between defatted balanites aeqyptiaca protein meal, protein concentrate and incidences of diabetes. This work investigated the anti- diabetic effects of balanites aeqyptiaca defatted protein meal and concentrate supplemented diets in streptozotocin (STZ)-induced diabetic rats. The rats were fattened for two weeks with high fat diet (HFD) to introduce Hyperglycemia and then made diabetic by intraperitoneal administration of STZ (35 mg/kg body weight) and fed diets containing 5 % defatted balanites aeqyptiaca protein meal (DAPM) and 5 % balanites aeqyptiaca protein concentrate (APC) for 14 days. The effect of the diet on blood glucose, serum glutathione peroxidase (GPx), glutathione transferase (GSH), thiobarbituric acid reactive species (TBARS), α-amylase and intestinal α-glucosidase activities were investigated. There was marked increase in the blood glucose, TBARS, pancreatic α-amylase and intestinal α-glycosidase with corresponding decrease in serum GPx and GSH contents in diabetic rats control groups. These trends were however, reversed in diabetic rats fed diet supplemented with the balanites aeqyptiaca protein meals for 14 days. The meals from defatted and protein concentrate inhibit α-amylase and α-glycosidase inhibitory activity in vivo. Thus, the anti-diabetes properties of the defatted meal and protein concentrate may be attributed to the influence of its constituent phytochemicals on starch digestion as well as endogenous enzymes activities. The study revealed that defatted aduwa meal and proteins concnentrate demonstrated potentials used as functional ingredients in food materials and could also increase income access of low resource populace.


Assuntos
Balanites , Diabetes Mellitus Experimental , Proteínas de Vegetais Comestíveis , Animais , Balanites/química , Glicemia/metabolismo , Diabetes Mellitus Experimental/dietoterapia , Diabetes Mellitus Experimental/metabolismo , Dieta , Glutationa Peroxidase/metabolismo , Hipoglicemiantes/farmacologia , Proteínas de Vegetais Comestíveis/farmacologia , Proteínas de Vegetais Comestíveis/uso terapêutico , Ratos , Ratos Wistar , Estreptozocina , Substâncias Reativas com Ácido Tiobarbitúrico/análise , alfa-Amilases/metabolismo
20.
BMC Genomics ; 23(1): 601, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35982422

RESUMO

BACKGROUND: Due to the prolonged usage of Bt-based biopesticides and Bt-transgenic crops worldwide, insects are continually developing resistance against Cry toxins. This resistance may occur if any mechanistic step in the insecticidal process is disrupted possibly because of the alteration in Cry-receptor binding affinity due to mutation in receptor genes. Compared to other lepidopteran insects, Cry receptor-related research has made asymmetric progress in the model insect Galleria mellonella. RESULTS: Present study describes the molecular characterization and functional analysis of five Cry toxin receptor-related genes (prohibitin, GLTP, α-amylase, ADAM and UDP-GT) and a gut repair gene (arylphorin) from the gut tissues of G. mellonella. Protein-protein docking analysis revealed that Cry1AcF putatively binds with all the five candidate proteins, suggesting their receptor-like function. These receptor-like genes were significantly overexpressed in the gut tissues of fourth-instar G. mellonella larvae upon early exposure to a sub-lethal dose of Cry1AcF toxin. However, targeted knockdown (by using bacterially-expressed dsRNAs) of these genes led to variable effect on insect susceptibility to Cry1AcF toxin. Insects pre-treated with prohibitin and α-amylase dsRNA exhibited significant reduction in Cry1AcF-induced mortality, suggesting their probable role as Cry receptor. By contrast, insects pre-treated with GLTP, ADAM and UDP-GT dsRNA exhibited no significant decline in mortality. This maybe explained by the possibility of RNAi feedback regulation (as few of the receptors belong to multigene family) or redundant role of GLTP, ADAM and UDP-GT in Cry intoxication process. CONCLUSION: Since the laboratory culture of G. mellonella develop Bt resistance quite rapidly, findings of the current investigation may provide some useful information for future Cry receptor-related research in the model insect.


Assuntos
Bacillus thuringiensis , Mariposas , Animais , Proteínas de Bactérias/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Larva/genética , Mariposas/genética , Mariposas/metabolismo , Proibitinas , Interferência de RNA , Difosfato de Uridina/metabolismo , Difosfato de Uridina/farmacologia , alfa-Amilases/genética , alfa-Amilases/metabolismo , alfa-Amilases/farmacologia
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