Ultrasonic-assisted enzymatic improvement of polyphenol content, antioxidant potential, and in vitro inhibitory effect on digestive enzymes of Miang extracts.

The aims of this research were to optimize the ultrasonic-assisted enzymatic extraction of polyphenols under Miang and tannase treatment conditions for the improvement of antioxidant activity of Miang extracts via response surface methodology. Miang extracts treated with and without tannase were investigated for their inhibitory effects on digestive enzymes. The optimal conditions for ultrasonic-assisted enzymatic extraction of the highest total polyphenol (TP) (136.91 mg GAE/g dw) and total flavonoid (TF) (5.38 mg QE/g dw) contents were as follows: 1 U/g cellulase, 1 U/g xylanase, 1 U/g pectinase, temperature (74 °C), and time (45 min). The antioxidant activity of this extract was enhanced by the addition of tannase obtained from Sporidiobolus ruineniae A45.2 undergoing ultrasonic treatment and under optimal conditions (360 mU/g dw, 51 °C for 25 min). The ultrasonic-assisted enzymatic extraction selectively promoted the extraction of gallated catechins from Miang. Tannase treatment improved the ABTS and DPPH radical scavenging activities of untreated Miang extracts by 1.3 times. The treated Miang extracts possessed higher IC50 values for porcine pancreatic α-amylase inhibitory activity than those that were untreated. However, it expressed approximately 3 times lower IC50 values for porcine pancreatic lipase (PPL) inhibitory activity indicating a marked improvement in inhibitory activity. The molecular docking results support the contention that epigallocatechin, epicatechin, and catechin obtained via the biotransformation of the Miang extracts played a crucial role in the inhibitory activity of PPL. Overall, the tannase treated Miang extract could serve as a functional food and beneficial ingredient in medicinal products developed for obesity prevention.

Safety and Effects of Lactobacillus paracasei TISTR 2593 Supplementation on Improving Cholesterol Metabolism and Atherosclerosis-Related Parameters in Subjects with Hypercholesterolemia: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Probiotics have the potential as a multi-target approach to modulate hypercholesterolemia associated with premature atherosclerosis. Various strains of Lactobacillus paracasei have been reported to affect hypercholesterolemia positively. This study aimed to investigate the effects of L. paracasei TISTR 2593 on lipid profile, cholesterol metabolism, and atherosclerosis according to the registration of Thai Clinical Trial Registry as identification number TCTR 20220917002. A total of 50 participants with hypercholesterolemia were randomly and equally assigned to consume L. paracasei TISTR 2593 or a placebo in maltodextrin capsules daily. Biomarkers of lipid profiles, oxidative stress state, inflammatory state, and other biological indicators were examined on days 0, 45, and 90. The results showed that subjects taking the L. paracasei TISTR 2593 could significantly reduce the level of serum low-density lipoprotein-cholesterol (p < 0.05), malondialdehyde (p < 0.001), and tumor necrosis factor-α (p < 0.01). Moreover, L. paracasei TISTR 2593 increased the level of serum apolipoprotein E (p < 0.01) and adiponectin (p < 0.001) significantly. No changes in serum total cholesterol, high-density lipoprotein-cholesterol, triglyceride, total bile acids, and monocyte chemoattractant protein-1 were observed during L. paracasei TISTR 2593 supplementation. Therefore, L. paracasei TISTR 2593 could be an adjuvant probiotic supplement to ameliorate hypercholesterolemia and prevent or delay the development of atherosclerosis.

Discovery of Natural Bisbenzylisoquinoline Analogs from the Library of Thai Traditional Plants as SARS-CoV-2 3CLPro Inhibitors: In Silico Molecular Docking, Molecular Dynamics, and In Vitro Enzymatic Activity.

The emergence of SARS-CoV-2 in December 2019 has become a global issue due to the continuous upsurge in patients and the lack of drug efficacy for treatment. SARS-CoV-2 3CLPro is one of the most intriguing biomolecular targets among scientists worldwide for developing antiviral drugs due to its relevance in viral replication and transcription. Herein, we utilized computer-assisted drug screening to investigate 326 natural products from Thai traditional plants using structure-based virtual screening against SARS-CoV-2 3CLPro. Following the virtual screening, the top 15 compounds based on binding energy and their interactions with key amino acid Cys145 were obtained. Subsequently, they were further evaluated for protein-ligand complex stability via molecular dynamics simulation and binding free energy calculation using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches. Following drug-likeness and ADME/Tox assessments, seven bisbenzylisoquinolines were obtained, including neferine (3), liensinine (4), isoliensinine (5), dinklacorine (8), tiliacorinine (13), 2'-nortiliacorinine (14), and yanangcorinine (15). These compounds computationally showed a higher binding affinity than native N3 and GC-373 inhibitors and attained stable interactions on the active site of 3CLpro during 100 ns in molecular dynamics (MD) simulation. Moreover, the in vitro enzymatic assay showed that most bisbenzylisoquinolines could experimentally inhibit SARS-CoV-2 3CLPro. To our delight, isoliensinine (5) isolated from Nelumbo nucifera demonstrated the highest inhibition of protease activity with the IC50 value of 29.93 µM with low toxicity on Vero cells. Our findings suggested that bisbenzylisoquinoline scaffolds could be potentially used as an in vivo model for the development of effective anti-SARS-CoV-2 drugs.

Brazilin from Caesalpinia sappan inhibits viral infection against PRRSV via CD163ΔSRCR5 MARC-145 cells: an in silico and in vitro studies.

This research aimed to identify bioactive compounds from Caesalpinia sappan extract that function as novel porcine reproductive and respiratory syndrome virus (PRRSV) infection inhibitors by computational molecular screening. We obtained a set of small-molecule compounds predicted to target the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. In addition, the functions of positive hits were assessed and verified utilizing an in vitro antiviral activity assay with PRRSV-infected MARC-145 cells. Combining molecular docking with the results of binding affinity and ligand conformation, it was found that brazilin had the highest binding energy with the SRCR5 receptor compared to catechin and epicatechin (- 5.8, - 5.5, and - 5.1 kcal/mol, respectively). In terms of molecular mechanics, the binding free energy between the SRCR5 receptor was - 15.71 kcal/mol based on the Poisson-Boltzmann surface area of brazilin. In addition, PRRSV infection in MARC-145 cells was significantly inhibited by brazilin compared to the control (virus titer, 4.10 vs. 9.25 TCID50/mL, respectively). Moreover, brazilin successfully limited the number of PRRSV RNA copies in MARC-145 cells as determined by RT-qPCR. By inhibiting the PRRSV-CD163 interaction with brazilin from Caesalpinia sappan, it may be possible to prevent PRRSV infection in pigs, as suggested by this research.

Decoding molecular recognition of inhibitors targeting HDAC2 via molecular dynamics simulations and configurational entropy estimation.

Molecular recognition by enzymes is a complicated process involving thermodynamic energies governing protein-ligand interactions. In order to aid the estimation of inhibitory activity of compounds targeting an enzyme, several computational methods can be employed to dissect this intermolecular contact. Herein, we report a structural dynamics investigation of an epigenetic enzyme HDAC2 in differentiating its binding to various inhibitors within the sub-sites of its active site. Molecular dynamics (MD) simulation was employed to elucidate the intermolecular interactions as well as the dynamics behavior of ligand binding. MD trajectories of five distinct HDAC2-inhibitor complexes reveal that compounds lacking adequate contacts with the opening rim of the active site possess high fluctuation along the cap portion, thus weakening the overall affinity. Key intermolecular interactions determining the effective binding of inhibitors include hydrogen bonds with Gly154, Asp181, and Tyr308; hydrophobic interactions between Phe155/Phe210 and the linker region; and a pi-stacking with Arg39 at the foot pocket. Decomposition of the binding free energy calculated per-residue by MM/PBSA also indicates that the interactions within the internal foot pocket, especially with residues Met35, Leu144, Gly305, and Gly306, can contribute significantly to the ligand binding. Additionally, configurational entropy of the binding was estimated and compared to the scale of the binding free energy in order to assess its contribution to the binding and to differentiate various ligand partners. It was found that the levels of entropic contribution are comparable among a set of structurally similar carbamide ligands, while it is greatly different for the set of unrelated ligands, ranging from 2.75 to 16.38 kcal/mol for the five inhibitors examined. These findings exemplify the importance of assessing molecular dynamics as well as estimating the entropic contribution in evaluating the ligand binding mechanism.

Synthesis, cytotoxicity evaluation and molecular docking studies on 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone derivatives.

2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC, 1) was isolated from seeds of Syzygium nervosum A.Cunn. ex DC. exhibiting intriguing biological activities. Herein, thirty three DMC derivatives including 4'-O-monosubstituted-DMC (2), 7-O-acylated-4-hydroxycoumarin derivatives (3), stilbene-coumarin derivatives (4), 2',4'-disubstituted-DMC (5), and flavanone derivatives (6), were synthesised through acylation, alkylations, and sulfonylation. These semi-synthetic DMC derivatives were evaluated for in vitro cytotoxicity against six carcinoma cell lines. It was found that most derivatives exhibited higher cytotoxicity than DMC. In particular, 4'-O-caproylated-DMC (2b) and 4'-O-methylated-DMC (2g) displayed the strongest cytotoxicity against SH-SY5Y with IC50 values of 5.20 and 7.52 µM, respectively. Additionally, 4'-O-benzylated-DMC (2h) demonstrated the strongest cytotoxicity against A-549 and FaDu with IC50 values of 9.99 and 13.98 µM, respectively. Our structure-activity relationship (SAR) highlights the importance of 2'-OH and the derivatisation pattern of 4'-OH. Furthermore, molecular docking simulation studies shed further light on how these bioactive compounds interact with cyclin-dependent kinase 2 (CDK2).

Identification of bioactive peptide from Oreochromis niloticus skin gelatin.

Fish skin, one type of wastes generated from Nile tilapia processing, is still a good source of collagen and gelatin. Bioactive peptides can be obtained from Nile tilapia skin gelatin by trypsin digestion. Trypsin hydrolysate was subsequently purified by gel filtration chromatography. Trypsin A fraction showed the greatest reducing power (5.138 ± 1.060 µM trolox/mg peptide) among all hydrolysate fractions, while trypsin B fraction from gel filtration column was found to exhibit the best radical scavenging and angiotensin-I-converting enzyme (ACE) inhibitory activities 8.16 ± 2.18 µg trolox/mg peptide and 59.32 ± 9.97 % inhibition, respectively. The most active fraction was subjected to MALDI-TOF/TOF MS/MS. After annotation by Mascot sequence matching software (Matrix Science) with Ludwig NR Database, two peptide sequences were identified; GPEGPAGAR (MW 810.87 Da) and GETGPAGPAGAAGPAGPR (MW 1490.61 Da). The docking analysis suggested that the shape of the shorter peptide may be slightly more proper, to fit into the binding cleft of the ACE. However, the binding affinities calculated from the docking showed no significant difference between the two peptides. In good agreement with the in silico data, results from the in vitro ACE inhibitory activity with synthetic peptides also showed no significant difference. Both peptides are thus interesting novel candidates suitable for further development as ACE inhibitory and antioxidant agents from the natural source.

Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.