Epicatechin

Cell lysates were obtained from 3T3-L1 or HepG2 cells. Cells were scraped and lysed with M-PER lysis buffer. After centrifugation for 15 min at 16,000×g, the supernatant was obtained, and protein content was quantified using Bradford reagent. Before drug treatment, the samples were diluted to achieve a protein concentration of 1 mg/mL. Samples were treated with the Kaem or DMSO for 2 h at 25 °C and then incubated with pronase (5, 10, and 20 µg/mL) or distilled water for 10 min at 25 °C. After the reaction, SDS was added to the sample and the samples were heated at 100 °C. A portion of each sample was used for LC–MS/MS analysis. Sample preparation and proteome analysis were conducted as indicated in the previous publication⁶⁹ . For western blot analysis, VDAC1 or Na⁺K⁺ ATPase was used as an internal control. For the structure–activity-relationship (SAR) analysis, kaempferol (Sigma-Aldrich, 60010), Acacetin (Sigma-Aldrich, 00017), isosakuranetin (Sigma-Aldrich, PHL82569), Biochanin A (Sigma-Aldrich, D2016), (−)Epicatechin (Sigma-Aldrich, E4018), Genistein (Sigma-Aldrich, G6649) were used.

The collection of crystal (X-ray) structure of the enzymes [PDB: 3RX3 (aldose reductase), 3W37 (α-glucosidase), and 1DHK (α-amylase)] were from the RSCB Protein Data Bank (https://www.rcsb.org/ accessed on 12 December 2020). The UCSF Chimera software V1.14 was used in the preparation of the enzymes in readiness for docking [40 (link)], PubChem (https://pubchem.ncbi.nlm.nih.gov/ accessed on 15 December 2020) was used to retrieve the structures of the chromatogram-identified phenolic compounds (sinapic acid, cacticin, hyperoside, 1,3-dicaffeoxyl quinic acid, procyanidin, rutin, epicatechin, isorhamnetin-3-O-rutinoside, chlorogenic acid, myricetin and luteolin-7-O-beta-d-glucoside) and standards (acarbose and ranirestat) and optimization of their three-dimensional structures executed using Avogadro software as previously reported [41 (link)]. The optimized compounds (ligands) and the enzymes were subsequently subjected to molecular docking.
The docking of the prepared phenolic compounds and standards into binding pockets of the enzymes (α-amylase, α-glucosidase, and aldose reductase) was by Autodock Vina Plugin on Chimera V1.14. Judging by the docking scores, complexes identified to have the best pose for each compound were ranked, selected and further analyzed through 100 ns molecular dynamics simulation (MDS).
The MDS was achieved as recently reported [28 (link)], using the GPU (force fields) version obtainable in AMBER package, where the description of the system by FF18SB variant of the AMBER force field was carried out [42 (link)]. With the aid of Restrained Electrostatic Potential (RESP) and the General Amber Force Field (GAFF) methods of the ANTECHAMBER assisted with information on atomic partial charges for the compounds. Hydrogen atoms and Na+ and Cl- counter ions (to neutralize the system) were made possible with Leap module of AMBER 18. The residues were numbered 1–336, 913, and 496, respectively, for aldose reductase, α-glucosidase and α-amylase. The system in each case was then lowered implicitly within an orthorhombic box of TIP3P water molecules such that all atoms were within 8Å of any box edge. MDS total time carried-out were 100 ns. For each simulation, hydrogens atoms were constricted using the SHAKE algorithm. The step size of each simulation was 2 fs, and an SPFP precision model was used. The simulations align with the isobaric-isothermal ensemble (NPT), having randomized seeding, Berendsen barostat maintains 1 bar constant pressure, 2 ps pressure-coupling constant, 300 K temperature and Langevin thermostat with a collision frequency of 1.0 ps [43 (link)].
Using PTRAJ, the systems were subsequently saved, and each trajectory analyzed every 1 ps, and the RoG, RMSF, and RMSD were analyzed with CPPTRAJ module (AMBER 18 suit).
Molecular Mechanics/GB Surface Area method (MM/GBSA) was adopted to assess the free binding energy while comparison of the systems binding affinity followed afterwards [44 (link)]. Binding free energy was averaged over 100,000 snapshots extracted from the 100 ns trajectory. The ΔG for each system (enzyme, complex and phenolics) was estimated as earlier reported [45 (link)].

Food grade cellulase (within a pH range of 3.0 to 6.5 and a temperature range of 35 to 75 °C), xylanase (within a pH range of 4.0 to 9.0 and a temperature range of 25 to 75 °C), and pectinase (within a pH range of 3 to 6.5 and a temperature range of 35 to 75 °C) were purchased from Winovazyme (Beijing, China). Porcine pancreatic α-amylase (with an optimal temperature of 20 °C and an optimal pH of 7.4), porcine pancreatic lipase (with an optimal temperature of 37 °C and an optimal pH of 7), and lysozyme were purchased from Sigma Aldrich (St. Louis, MO, USA). Tannic acid, methyl gallate, gallic acid, rhodanine, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), potassium sulfate, quercetin, and Folin-Ciocalteu’s phenol reagent were all of analytical grade and of the highest quality available from Sigma-Aldrich. High-performance liquid chromatography (HPLC) grade standard (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC), (-)-gallocatechin gallate (GCG), (-)-catechin gallate (CG), (-)-gallocatechin (GC), (+)-catechin (C), caffeine, and gallic acid (GA) were all purchased from Sigma. All chemicals used for antioxidant activity assays and enzyme production were of analytical grade and were obtained from RCI Labscan (Bangkok, Thailand). The medium ingredients used in this study, such as agar, yeast extract, and malt extract, were all purchased from HiMedia (Nashik, India).

It is of the utmost importance that the quality and shelf stability of a meat product is maintained during its storage. It has been discovered that the addition of different kinds of fiber sources to meat products can alter the preservation quality in a variety of ways. The inclusion of chia seeds in (camburger) camel burger, too has recorded a reduced TBA (thiobarbituric acid) value, which implies lesser lipid oxidation in comparison to the control, and these burgers were determined to be organoleptically acceptable after storage for a period of 12 days (Zaki, 2018 (link)). Because it better inhibits oxymyoglobin oxidation, the addition of oat flour to chicken kofta has resulted in a product that is microbiologically safe and sensorial acceptable over the entire period of 15 days of storage (Lin & Lin, 2006 (link)). Grape seed, which includes flavonoids including catechin, epicatechin, procyanidins, and other chemicals with antimicrobial properties, is the most widely utilized and documented industrial waste for this purpose (Friedman, 2014 (link)). Uncertain processes, including disruption of the cytoplasmic membrane, blockage of specific metabolic pathways and enzymes, and chelation of critical metals for growth like zinc and iron, underlie the antibacterial effects of polyphenols and other natural chemicals (Daglia, 2012 (link)). Incorporation of grape seed in raw pork in aerobic packaging at 20 °C, when inoculated with 10⁵ CFU of Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica. Decreased growth of L. monocytogenes by 17.5%, S. aureus by 14%, and S. entericaby 20% (Shan et al., 2009 (link)). A 4-log decrease in the growth of L. monocytogenes at 4 °C, 1 log at 7 °C, and no changes at 12 °C was noticed in meat paté incorporated with pomegranate peel. The inoculation was done with 4 log CFU/g of L. monocytogenes at 4 °C, 7 °C, and 12 °C for 46 days (Hayrapetyan, Hazeleger & Beumer, 2012 (link)). A few other examples have been listed in Table 2, indicating the shelf-life through changes in microbiological characteristics and antioxidant capacity of different meat products as influenced by various incorporations.