Preparation of a Novel Oat ß-Glucan-Chromium(III) Complex and Its Hypoglycemic Effect and Mechanism.

This study synthesized a novel oat ß-glucan (OBG)-Cr(III) complex (OBG-Cr(III)) and explored its structure, inhibitory effects on α-amylase and α-glucosidase, and hypoglycemic activities and mechanism in vitro using an insulin-resistant HepG2 (IR-HepG2) cell model. The Cr(III) content in the complex was found to be 10.87%. The molecular weight of OBG-Cr(III) was determined to be 7.736 × 104 Da with chromium ions binding to the hydroxyl groups of OBG. This binding resulted in the increased asymmetry and altered spatial conformation of the complex along with significant changes in morphology and crystallinity. Our findings demonstrated that OBG-Cr(III) exhibited inhibitory effects on α-amylase and α-glucosidase. Furthermore, OBG-Cr(III) enhanced the insulin sensitivity of IR-HepG2 cells, promoting glucose uptake and metabolism more efficiently than OBG alone. The underlying mechanism of its hypoglycemic effect involved the modulation of the c-Cbl/PI3K/AKT/GLUT4 signaling pathway, as revealed by Western blot analysis. This research not only broadened the applications of OBG but also positioned OBG-Cr(III) as a promising Cr(III) supplement with enhanced hypoglycemic benefits.

The antihyperglycemic potential of pyrazolobenzothiazine 1, 1-dioxide novel derivative in mice using integrated molecular pharmacological approach.

Diabetes Mellitus is a metabolic disease characterized by elevated blood sugar levels caused by inadequate insulin production, which subsequently leads to hyperglycemia. This study was aimed to investigate the antidiabetic potential of pyrazolobenzothiazine derivatives in silico, in vitro, and in vivo. Molecular docking of pyrazolobenzothiazine derivatives was performed against α-glucosidase and α-amylase and compounds were selected based on docking score, bonding interactions and low root mean square deviation (RMSD). Enzyme inhibition assay against α-glucosidase and α-amylase was performed in vitro using p-nitrophenyl-α-D-glucopyranoside (PNPG) and starch substrate. Synthetic compound pyrazolobenzothiazine (S1) exhibited minimal conformational changes during the 100 ns MD simulation run. S1 also revealed effective IC50 values for α-glucosidase (3.91 µM) and α-amylase (8.89 µM) and an enzyme kinetic study showed low ki (- 0.186 µM, - 1.267 µM) and ki' (- 0.691 µM, - 1.78 µM) values with the competitive type of inhibition for both enzymes α-glucosidase and α-amylase, respectively. Moreover, studies were conducted to check the effect of the synthetic compound in a mouse model. A low necrosis rate was observed in the liver, kidney, and pancreas through histology analysis performed on mice. Compound S1 also exhibited a good biochemical profile with lower sugar level (110-115 mg/dL), increased insulin level (25-30 µM/L), and low level of cholesterol (85 mg/dL) and creatinine (0.6 mg/dL) in blood. The treated mice group also exhibited a low % of glycated haemoglobin (3%). This study concludes that S1 is a new antidiabetic-agent that helps lower blood glucose levels and minimizes the complications associated with type-II diabetes.

Enzymatic Assembly of Chitosan-Based Network Polysaccharides and Their Encapsulation and Release of Fluorescent Dye.

We prepared network polysaccharide nanoscopic hydrogels by crosslinking water-soluble chitosan (WSCS) with a carboxylate-terminated maltooligosaccharide crosslinker via condensation. In this study, the enzymatic elongation of amylose chains on chitosan-based network polysaccharides by glucan phosphorylase (GP) catalysis was performed to obtain assembly materials. Maltoheptaose (Glc7) primers for GP-catalyzed enzymatic polymerization were first introduced into WSCS by reductive amination. Crosslinking of the product with the above-mentioned crosslinker by condensation was then performed to produce Glc7-modified network polysaccharides. The GP-catalyzed enzymatic polymerization of the α-d-glucose 1-phosphate monomer from the Glc7 primers on the network polysaccharides was conducted, where the elongated amylose chains formed double helices. Enzymatic disintegration of the resulting network polysaccharide assembly successfully occurred by α-amylase-catalyzed hydrolysis of the double helical amyloses. The encapsulation and release of a fluorescent dye, Rhodamine B, using the CS-based network polysaccharides were also achieved by means of the above two enzymatic approaches.

Influences of superfine-grinding and enzymolysis separately assisted with carboxymethylation and acetylation on the in vitro hypoglycemic and antioxidant activities of oil palm kernel expeller fibre.

The synergistic effects of ultrafine grinding and enzymolysis (cellulase and Laccase hydrolysis) alone or combined with carboxymethylation or acetylation on the hypoglycemic and antioxidant activities of oil palm kernel fibre (OPKEF) were studied for the first time. After these synergistic modifications, the microstructure of OPKEF became more porous, and its soluble fibre and total polyphenols contents, and surface area were all improved (P < 0.05). Superfine-grinding and enzymolysis combined with carboxymethylation treated OPKEF exhibited the highest viscosity (13.9 mPa∙s), inhibition ability to glucose diffusion (38.18%), and water-expansion volume (3.58 mL∙g-1). OPKEF treated with superfine-grinding and enzymolysis combined with acetylation showed the highest surface hydrophobicity (50.93) and glucose adsorption capacity (4.53 µmol∙g-1), but a lower α-amylase-inhibition ability. Moreover, OPKEF modified by superfine-grinding and enzymolysis had the highest inhibiting activity against α-amylase (25.78%). Additionally, superfine-grinding and enzymolysis combined with carboxymethylation or acetylation both improved the content and antioxidant activity of OPEKF's bounding polyphenols (P < 0.05).

Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcusabyssi.

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The Km value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding Vmax value was 25.00 ± 0.67 µmol min-1 mg-1 in the presence of 2 mM CaCl2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications.

High-confidence structural annotation of substances via multi-layer molecular network reveals the system-wide constituent alternations in milk interfered with diphenylolpropane.

The spectral database-based mass spectrometry (MS) matching strategy is versatile for structural annotating in ingredient fluctuation profiling mediated by external interferences. However, the systematic variability of MS pool attributable to aliasing peaks and inadequacy of present spectral database resulted in a substantial metabolic feature depletion. An amended procedure termed multiple-charges overlap peaks extraction algorithm (MCOP) was proposed involving identifying collision-trigged dissociation precursor ions through iteratively matching mass features of fragmentations to expand the spectral reference library. We showcased the versatility and utility of established strategy in an investigation centered on the stimulation of milk mediated by diphenylolpropane (BPA). MCOP enabled efficient unknown annotations at metabolite-lipid-protein level, which elevated the accuracy of substance annotation to 85.3% after manual validation. Arginase and α-amylase (|r| > 0.75, p < 0.05) were first identified as the crucial issues via graph neural network-based virtual screening in the abnormal metabolism of urea triggered by BPA, resulting in the accumulation of arginine (original: 1.7 µg kg-1 1.7 times) and maltodextrin (original: 6.9 µg kg-1 2.9 times) and thus, exciting the potential dietary risks. Conclusively, MCOP demonstrated generalisation and scalability and substantially advanced the discovery of unknown metabolites for complex matrix samples, thus deciphering dark matter in multi-omics.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Simultaneous improvement of thermostability and maltotriose-forming ability of a fungal α-amylase for bread making by directed evolution.

For applications in food industries, a fungal α-amylase from Malbranchea cinnamomea was engineered by directed evolution. Through two rounds of screening, a mutant α-amylase (mMcAmyA) was obtained with higher optimal temperature (70 °C, 5 °C increase) and better hydrolysis properties (18.6 % maltotriose yield, 2.5-fold increase) compared to the wild-type α-amylase (McAmyA). Site-directed mutations revealed that Threonine (Thr) 226 Serine (Ser) substitution was the main reason for the property evolution of mMcAmyA. Through high cell density fermentation, the highest expression level of Thr226Ser was 3951 U/mL. Thr226Ser was further used for bread baking with a dosage of 1000 U/kg flour, resulting in a 17.8 % increase in specific volume and a 35.6 % decrease in hardness compared to the control. The results were a significant improvement on those of McAmyA. Moreover, the mutant showed better anti-staling properties compared to McAmyA, as indicated by the improved sensory evaluation after 4 days of storage at 4 and 25 °C. These findings provide insights into the structure-function relationship of fungal α-amylase and introduce a potential candidate for bread-making industry.

Design, synthesis, α-amylase and glucose diffusion inhibition, and molecular docking studies of new indenopyrazolones bearing benzothiazole derivatives.

An eco-friendly facile synthesis of a series of twenty 1-(4/6-substitutedbenzo[d]thiazol-2-yl)-3-(phenyl/substitutedphenyl)indeno[1,2-c]pyrazol-4(1H)-ones 7a-t was achieved by the reaction of 2-(benzoyl/substitutedbenzoyl)-(1H)-indene-1,3(2H)-dione 3a-t and 2-hydrazinyl-4/6-substitutedbenzo[d]thiazole 6a-t in presence of freshly dried ethanol and glacial acetic acid under reflux conditions in good yields. The newly synthesized derivatives were well characterized using different physical and spectral techniques (FTIR, 1H NMR & 13C NMR, and HRMS). All the compounds were subjected to assess their in vitro α-amylase and glucose diffusion inhibitory activity. Amongst them, the compounds 7i and 7l showed better α-amylase inhibitory activity demonstrating IC50 values of 92.99±1.94 µg/mL and 95.41±3.92 µg/mL, respectively in comparison to the standard drug acarbose (IC50 value of 103.60±2.15 µg/mL). The derivatives 7d and 7k exhibited good glucose diffusion inhibition with values of 2.25±1.16 µg/mL and 2.63±1.45 µg/mL, respectively with standard reference acarbose (2.76±0.55 µg/mL). The observed α-amylase inhibitory activity findings were corroborated through molecular docking investigations, particularly for the highly active compounds 7i (binding energy -8.0 kcal/mol) and 7l (binding energy -8.2 kcal/mol) respectively, in comparison to acarbose with a value of binding energy -6.9 kcal/mol for α-amylase.

Studies on α-amylase inhibition by acarbose and quercetin using fluorescence, circular dichroism, docking, and dynamics simulations.

This study looked at the effects of acarbose (ACA) and quercetin (QUE) on α-amylase activity, employing QUE and ACA to measure enzyme activity. The study observed that both drugs suppressed α-amylase activity, with greater inhibition reported at higher concentrations. The use of tryptophan residues as an intrinsic fluorescence probe permitted the observation of conformational changes in α-amylase, with CD measurements utilized to explore the secondary structure in the presence of QUE and ACA. Docking studies revealed an effective interaction between α-amylase, quercetin and acarbose, with a higher binding energy. Finally, a trajectory analysis was done to establish the stability and volatility of these complexes. These findings have potential significance for the development of new α-amylase-related therapeutics.

Pain and salivary biomarkers of stress in temperomandibular disorders were affected by maxillary splints.

BACKGROUND: Longitudinal intervention studies on treatment options in temporomandibular dysfunction (TMD) including self reports and salivary biomarkers of stress are rare and the exact therapeutic function of occlusal splints widely unknown. METHODS: We examined the therapeutic effects of a Michigan splint with occlusal relevance in patients with TMD using a placebo-controlled, delayed-start design. Two intervention groups received a Michigan splint, while one of them had a placebo palatine splint for the first 3 weeks. We collected pain intensities (at rest and after five occlusal movements), salivary measures associated with stress (cortisol and alpha-amylase) and self-reported psychological distress (stress, anxiety, catastrophizing) at baseline and 3 and 7 weeks after onset of intervention. RESULTS: At baseline, we observed increased pain intensity and psychological distress in TMD patients compared to 11 matched healthy controls. Baseline anxiety was linked to movement pain intensity through stress. Over therapy reductions in pain intensity and morning cortisol were more pronounced in those patients starting immediately with the Michigan splint, while psychological distress decreased similarly in both groups. CONCLUSION: Our results suggest that perceived stress plays a role for the association between anxiety and TMD pain and underlines the need for an interdisciplinary perspective on the pathogenesis and therapy of TMD in a setting where psychotherapeutic knowledge is still scarce or rarely applied.

Enhancing the anti-thixotropic properties of waxy maize starch modified by different α-amylases and its underlying molecular mechanism.

The large thixotropy of the starch-thickened foods is often unfavorable in many applications. This study examined the contribution of the proportion of amylopectin chain length to time-dependence of starch gels. The α-amylase (AM) from Bacillus stearothermophilus and maltogenic α-amylase (MA) from Bacillus subtilis were used to trim amylopectin in different reaction patterns. HPLC, HPAEC and IBC data suggested AM attacked B-chains (DP 12-36), causing an increment in number of the chains with DP 6-12, whereas MA primarily trimmed the short B-chains (DP 12-18) and partial A-chains (DP 9-12) to generate short chains with DP 6-9. Interestingly, the recovery of AM-gels was faster than MA-gels at the same degree of hydrolysis when subjected to shear according to the linear correlation analysis. When releasing the same mass of sugar, shortening of the long internal chains played an important role in reducing time dependence of starch gel rather than the external side chains. Possible models were proposed to illustrate the differences in the mechanism of rapid-recovery caused by different side-chain distributions. The outcome provided a new perspective to regulate the thixotropy behavior of starch through enzyme strategies in the granular state.

Production of polyhydroxyalkanoates by engineered Halomonas bluephagenesis using starch as a carbon source.

A novel α-amylase Amy03713 was screened and cloned from the starch utilization strain Vibrio alginolyticus LHF01. When heterologously expressed in Escherichia coli, Amy03713 exhibited the highest enzyme activity at 45 °C and pH 7, maintained >50 % of the enzyme activity in the range of 25-75 °C and pH 5-9, and sustained >80 % of the enzyme activity in 25 % (w/v) of NaCl solution, thus showing a wide range of adapted temperatures, pH, and salt concentrations. Halomonas bluephagenesis harboring amy03713 gene was able to directly utilize starch. With optimized amylase expression, H. bluephagenesis could produce poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB). When cultured for PHB production, recombinant H. bluephagenesis was able to grow up to a cell dry weight of 11.26 g/L, achieving a PHB titer of 6.32 g/L, which is the highest titer that has been reported for PHB production from starch in shake flasks. This study suggests that Amy03713 is an ideal amylase for PHA production using starch as the carbon source in H. bluephagenesis.

Comparative analysis of granular starch hydrolysis and multi-structural changes by diverse α-amylases sources: Insights from waxy rice starch.

Three cultivars of waxy rice starch with different multi-scale structures were subjected to α-amylase hydrolysis to determine amylopectin fine structure, production of oligosaccharides, morphology, and crystallinity of the partially hydrolyzed starch granules. α-amylases hydrolyzed the amylopectin B2 chain during the initial stage of hydrolysis, suggesting that it is primarily located in the outer shell of the granules. For waxy rice starch with loose structure, α-amylases attacked the crystalline and amorphous regions simultaneously in the initial stage, while for starch granules with compact structure, the outer shell blocklet (crystalline structure) can be a hurdle for α-amylases to proceed to hydrolysis of the internal granule structure. The ability of α-amylases from porcine pancreatic α-amylases to attack the outer shell crystalline structure was lower than that of α-amylases from Bacillus amyloliquefaciens and Aspergillus oryzae. These results show that α-amylase source and rice cultivar combinations can be used to generate diverse structures in degraded waxy rice starch.

In vitro inhibitory effect of five natural sweeteners on α-glucosidase and α-amylase.

A promising and efficacious approach to manage diabetes is inhibiting α-glucosidase and α-amylase activity. Therefore, the inhibitory activities of five natural sweeteners (mogrosides (Mog), stevioside (Ste), glycyrrhizinic acid (GA), crude trilobatin (CT), and crude rubusoside (CR)) against α-glucosidase and α-amylase and their interactions were evaluated in vitro using enzyme kinetics, fluorescence spectroscopy, Fourier infrared spectroscopy, and molecular docking. The inhibitor sequence was CT > GA > Ste, as GA competitively inhibited α-glycosidase activity while CT and Ste exhibited mixed inhibitory effects. Compared to a positive control acarbose, the inhibitory activity of CT was higher. For α-amylase, the mixed inhibitors CT, CR, and Mog and the competitive inhibitor Ste effectively inhibited the enzyme, with the following order: CT > CR > Ste > Mog; nevertheless, the inhibitors were slightly inferior to acarbose. Three-dimensional fluorescence spectra depicted that GA, CT, and CR bound to the hydrophobic cavity of α-glucosidase or α-amylase and changed the polarity of the hydrophobic amino acid-based microenvironment and structure of the polypeptide chain backbone. Infrared spectroscopy revealed that GA, CT, and CR could disrupt the secondary structure of α-glucosidase or α-amylase, which decreased enzyme activity. GA, trilobatin and rubusoside bound to amino acid residues through hydrogen bonds and hydrophobic interactions, changing the conformation of enzyme molecules to decrease the enzymatic activity. Thus, CT, CR and GA exhibit promising inhibitory effects against α-glucosidase and α-amylase.

Indenoquinoxaline-phenylacrylohydrazide hybrids as promising drug candidates for the treatment of type 2 diabetes: In vitro and in silico evaluation of enzyme inhibition and antioxidant activity.

Existing drugs that are being used to treat type-2 diabetes mellitus are associated with several side effects; thus, exploring potential drug candidates is still an utter need these days. Hybrids of indenoquinoxaline and hydrazide have never been explored as antidiabetic agents. In this study, a series of new indenoquinoxaline-phenylacrylohydrazide hybrids (1-30) were synthesized, structurally characterized, and evaluated for α-amylase and α-glucosidase inhibitory activities, as well as for their antioxidant properties. All scaffolds exhibited varying degrees of inhibitory activity against both enzymes, with IC50 values ranging from 2.34 to 61.12 µM for α-amylase and 0.42 to 54.72 µM for α-glucosidase. Particularly, compounds 10, 16, 17, 18, 24, and 25 demonstrated the highest efficacy in inhibiting α-amylase, while compounds 6, 7, 8, 10, 12, 14, 13, 16, 17, 18, 24, and 25 were the most effective α-glucosidase inhibitors, compared to standard acarbose. Moreover, most of these compounds displayed substantial antioxidant potential compared to standard butylated hydroxytoluene (BHT). Kinetics studies revealed competitive inhibition modes by compounds. Furthermore, a comprehensive in silico study and toxicity prediction were also conducted, further validating these analogs as potential drug candidates. The structured compounds demonstrated enhanced profiles, underscoring their potential as primary candidates in drug discovery.

Study on the preparation and enzyme inhibitory activity of polyphenols from Sargassum pallidum.

This study aimed to obtain a high yield and purity of Sargassum pallidum polyphenol extracts (SPPE) and study its enzyme activity. Fresh Sargassum pallidum seaweed was selected for optimization of ultrasound-assisted extraction (UAE) conditions and purification conditions using macroporous resin and Sephadex LH20 to obtain SPPE. The SPPE was characterized using UPLC-QTOF-MS/MS and α-amylase, α-glucosidase, tyrosinase, and AchE inhibitory activity were determined. The maximum extraction rate of SPPE was 7.56 mg GAE/g and the polyphenol purity reached 70.5% after macroporous resin and Sephadex LH-20 purification. A total of 50 compounds were identified by UPLC-QTOF-MS/MS. The IC50 values of SPPE were 334.9 µg/mL, 6.290 µg /mL, 0.834 mg /mL and 0.6538 mg /mL for α-amylase, α-glucosidase, tyrosinase and AchE, respectively. Molecular docking technology further revealed the effects of SPPE on the above enzymes. This study provided information on the potential hypoglycemic, whitening and anti-Alzheimer's disease biological activities of SPPE, which had guiding significance for the purification and development of other seaweed polyphenols.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2.

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1-3% H2O2.

PpAmy1 Plays a Role in Fruit-Cracking by Regulating Mesocarp Starch Hydrolysis of Nectarines.

Nectarine [Prunus persica (L.) Batsch var.] fruits are highly susceptible to cracking during the ripening process, which significantly decreases their commercial value. In this study, we investigated the underlying mechanism of nectarine fruit-cracking using two nectarine varieties, namely, "Qiannianhong" (cracking-susceptible) and "CR1012" (cracking-resistant). Our findings indicate that nectarine fruit-cracking occurs during the second stage of fruit expansion. Despite no differences in epicarp cell size between "Qiannianhong" and "CR1012", the mesocarp cells of "Qiannianhong" were larger than those of "CR1012". Moreover, a comparison of starch hydrolysis between the two varieties revealed that "CR1012" had higher starch content in the mesocarp but lower soluble sugar content compared to "Qiannianhong". Additionally, by testing the α-amylase and ß-amylase activity of the mesocarp, our results showed a difference only in α-amylase activity between the two varieties. Furthermore, qRT-PCR detection indicated a higher expression level of the PpAmy1 (α-amylase synthesis gene) in "Qiannianhong" compared to "CR1012". To further investigate the role of PpAmy1, we employed RNAi technology to suppress its expression in "Qiannianhong" fruits. The results showed a significant reduction in α-amylase activity, starch hydrolysis, soluble sugar content, cell size of the mesocarp, and fruit-cracking. These findings underscore the pivotal role of PpAmy1 in the occurrence of nectarine fruit cracking.

Synthesis, structural characterizations, in vitro biological evaluation and computational investigations of pyrazole derivatives as potential antidiabetic and antioxidant agents.

In this study, a two pyrazole derivatives; 2-(5-methyl-1H-pyrazole-3-carbonyl)-N-phenylhydrazine-1-carboxamide (Pyz-1) and 4-amino-5-(5-methyl-1H-pyrazol-3-yl)-4H-1,2,4-triazole-3-thiol (Pyz-2) were synthesized and characterized by 13C-NMR, 1H-NMR, FT-IR, and mass spectrometry. A complete molecular structures optimization, electronic and thermodynamic properties of Pyz-1 and Pyz-2 in gas phase and aqueous solution were predicted by using hybrid B3LYP method with the 6-311++G** basis sets. Pyz-1 and Pyz-2 were evaluated in vitro for their anti-diabetic, antioxidant and xanthine oxidase inhibition activities. For anti-diabetic activity, Pyz-1 and Pyz-2 showed a potent α-glucosidase and α-amylase inhibition with IC50 values of 75.62 ± 0.56, 95.85 ± 0.92 and 119.3 ± 0.75, 120.2 ± 0.68 µM, respectively, compared to Acarbose (IC50(α-glucosidase) = 72.58 ± 0.68 µM, IC50(α-amylase) = 115.6 ± 0.574 µM). In xanthine oxidase assay, Pyz-1 and Pyz-2 exhibited remarkable inhibitory ability with IC50 values 24.32 ± 0.78 and 10.75 ± 0.54 µM, respectively. The result of antioxidant activities showed that the title compounds have considerable antioxidant and radical scavenger abilities. In addition, molecular docking simulation was used to determine the binding modes and energies between the title compounds and α-glucosidase and α-amylase enzymes.