Binding interaction between (−)-epigallocatechin-3-gallate (EGCG) of green tea and pepsin

Analysis of the binding interaction of (−)-epigallocatechin-3-gallate (EGCG) and pepsin is important for understanding the inhibition of digestive enzymes by tea polyphenols. We studied the binding of EGCG to pepsin using fluorescence spectroscopy, Fourier transform infrared spectroscopy, isothermal titration calorimetry, and protein-ligand docking. We found that EGCG could inhibit pepsin activity. According to thermodynamic parameters, a negative ΔG indicated that the interaction between EGCG and pepsin was spontaneous, and the electrostatic force accompanied by hydrophobic binding forces may play major role in the binding. Data from multi-spectroscopy and docking studies suggest that EGCG could bind pepsin with a change in the native conformation of pepsin. Our results provide further understanding of the nature of the binding interactions between catechins and digestive enzymes

Analysis of Quercetin from Allium cepa L. Skin Waste in Inhibiting α-Amylase: An Alternate Therapeutic Drug to Treat Diabetes

Type 2 diabetes is a chronic metabolic disease characterized by an increase in blood sugar due to insulin resistance as well as the decline of insulin production by the pancreas. Quercetin is a flavonoid secondary metabolite that could be utilized as an α-amylase inhibitor and can potentially be used as a therapeutic drug in type 2 diabetes treatment by inhibiting starch hydrolysis in the human body. This research aims to determine the quercetin yield of Allium cepa L. skin waste, evaluate the α-amylase inhibitory properties of the extracted quercetin, and delve into the molecular mechanism of α-amylase inhibition by quercetin. Methods that were used in this research consisted of sample collection and preparation, quercetin extraction, testing for quercetin content in extract by spectrophotometry and HPLC, as well as testing for α-amylase inhibition by quercetin. Quercetin extraction produced 1.52 g of dry extract, yielding 16.8 mg of extract per g of dry shallot skin. Spectrophotometric analysis yielded 3.37 mg of quercetin per gram of dried shallot skin, while HPLC yielded 3.99 mg/g. The shallot skin extract inhibited α-amylase with an IC50 of 348.2 µg/mL. In comparison, acarbose had a more potent inhibitory effect, with an IC50 of 237.4 µg/mL. Quercetin inhibits α-amylase by forming hydrogen bonds with the active sites of the enzyme (Asp197, Glu233, and Asp300)

Encapsulation of bioactive nutraceutical compounds in donkey and bovine milk β-casein-based carriers

The use of self-assembled -caseins from bovine origin as nanocarriers for the delivery of nutraceutical compounds or drugs has been widely investigated. Concerning β-caseins from other milk sources, the use of hypoallergenic donkey β-CN as a potential delivery vehicle for nutraceutical hydrophobic compounds is beginning to generate interest. Therefore, the purpose of this study was to characterize the self-assembly properties and structural changes of purified β-CN obtained from hypoallergenic donkey milk under different conditions of pH, temperature, and ionic strength, with the comparison of commercial bovine β-CN. Based on this knowledge, the interaction mechanisms between donkey β-CN with vitamin D2, and resveratrol, were studied, as well as the encapsulation efficiency of resveratrol within β-CN micelles. These data can provide a theoretical basis for promoting the enrichment and bioavailability of hydrophobic bioactive compounds in food products. This thesis work has been structured as follows: CHAPTER 1 delves into the structure and attributes of casein micelles while highlighting bovine β-CN's favored role as an encapsulation material due to its unique structure. Intriguingly, similarities in self-assembly patterns between donkey and bovine β-CN were detected, seen through comparable CMC and CMT behaviors. It is interesting to further explore donkey milk-derived β-CN with hypoallergenic properties as nanocarriers for bioactive substances. Therefore, CHAPTER 2 examined the self-assembly, secondary structure, and surface hydrophobicity of isolated donkey β- CN under varying pH, temperature, and buffer concentration conditions, then was compared with commercial bovine β-CN. The results offer valuable insights into the factors that influence molecular interactions driving donkey β-CN self-association, which could be applied to the development of nanocarriers for bioactive compound encapsulation in pharmaceutical and nutraceutical contexts. Based on this concept, CHAPTER 3 studied the interaction mechanisms between both donkey and bovine β-CN, and vitamin D2 (VD2) were explored using fluorescence and Dynamic Laser Light Scattering (DLS) techniques at elevated vitamin/protein ratios. Furthermore, the mechanism of interaction between both β-CNs and resveratrol, as well as the ability of these protein micelles to encapsulate resveratrol are studied in CHAPTER 4. Lastly, CHAPTER 5 imparts the general discussion to declare the main findings of this thesis, and their potential applications in donkey β-CN encapsulating or delivering bioactive substances with the purpose of enhancing their stability and bioactivity

Perturbation of enzyme structure by nano-metal organic frameworks:A question mark on their safety-by-design?

Our study investigates the interactions between nanoscale Metal-Organic Frameworks (nMOFs), specifically ZIF-8 and CuIm, and key enzymes: Acetylcholine Esterase (AChE), α-amylase. Using circular dichroism (CD) spectroscopy, we observed significant alterations in the secondary structures of these enzymes upon interaction with nMOFs. AChE showed a reduction in α-helix content from 20.1 % to a significantly lower value when exposed to 160 µg/mL of nMOFs, with a corresponding increase in β-sheet and other structural components. Enzymatic activity assays revealed that CuIm nMOFs decreased AChE activity by 67.08 % at the highest concentration tested (160 µg/mL). ZIF-8 also affected AChE activity significantly at this concentration. Similarly, α-amylase exhibited structural changes, with increasing concentrations of nMOFs leading to a near-total loss of secondary structure at 80 and 160 µg/mL. These structural changes were accompanied by a marked decrease in enzymatic activity, particularly with CuIm nMOFs showing the most substantial inhibitory effects. Our findings highlight the profound impact of nMOFs on enzyme structures and functions, emphasising the need for comprehensive assessments of nMOFs' potential toxicity and understanding the aspects of their safety-by-design.</p

Biophysical characterization of human serum albumin interaction with dapagliflozin: multi-spectroscopic and molecular docking study

Human serum albumin (HSA) is the most abundant protein in human blood plasma and plays a crucial role in drug transport and pharmacokinetics. Dapagliflozin (DAPA), a sodium-glucose co-transporter 2 (SGLT2) inhibitor, is widely prescribed for the treatment of type 2 diabetes mellitus. In the present study, we employed a combination of multi-spectroscopic techniques, including fluorescence spectroscopy (three-dimensional, synchronous), UV-visible absorption spectroscopy, thermodynamic analysis, and molecular docking to investigate the interaction of dapagliflozin with HSA under physiological condition. The quenching mechanism of DAPA was determined to be dynamic through Stern-Volmer and modified Stern-Volmer analyses. The binding constants at 298 K, 303 K, 308 K were 0.52x104, 0.303x104 and 0.264x104 M-1, respectively. Thermodynamic analysis revealed that the binding process is spontaneous, driven primarily by hydrogen bonding and hydrophobic interactions at various temperatures. Synchronous fluorescence studies suggest that DAPA binding does not significantly alter the microenvironment around the tyrosine and tryptophan residues of HSA, implying that the binding sites are spatially distinct from these residues. Three-dimensional fluorescence studies reveal that the addition of DAPA to HSA affects changes in the micro-environment and conformation of HSA. UV-VIS spectroscopy confirmed the formation of the HSA-DAPA complex, characterized by spectral shifts in both peptide bond and aromatic amino acid regions, indicating alterations in the protein\u27s secondary structure. The decrease in zeta potential upon DAPA binding suggests a change in the surface charge and potential conformational changes in HSA, which may influence its biological activity and interaction with other molecules. Molecular docking studies identified key amino acid residues involved in the binding of DAPA to HSA, primarily through hydrophobic and hydrogen bond interactions

Comparison of antigenicity and conformational changes to β-lactoglobulin following kestose glycation reaction with and without dynamic high-pressure microfluidization treatment

Previous work indicated that conformational changes of β-lactoglobulin (β-LG) induced by dynamic high pressure microfluidization (DHPM) was related to the increase of antigenicity. In this study, β-LG glycated with 1-kestose and combined with DHPM decreased the antigenicity of β-LG. The antigenicity of control, β-LG-kestose (0.1 MPa) and β-LG-kestose (80 MPa) were 100, 79 and 42 μg/mL respectively. The molecular weight of β-LG conjugated to kestose increased from 18.4 to 19.6 kDa and its conformation scarcely changed. Conversely, combined with DHPM treatment (80 MPa), β-LG conjugated to kestose formed two conjugates with molecular weight of 18.8 and 19.8 kDa, respectively. Furthermore, the unfolding of β-LG as a result of the treatments is reflected by a decrease of intrinsic and synchronous fluorescence intensity and changes to the secondary structure. The conformational changes induced by DHPM and glycation treatments synergistically decrease the antigenicity of β-LG due to more masked or disrupted epitopes