Rational design of an N-terminal cysteine-containing tetrapeptide that inhibits tyrosinase and evaluation of its mechanism of action.

There has been a resurgence of interest in bioactive peptides as therapeutic agents. This is particularly interesting for tyrosinase, which can be inhibited by thiol-containing peptides. This work demonstrates that an N-terminal cysteine-containing tetrapeptide can be rationally designed to inhibit tyrosinase activity in vitro and in cells. The tetrapeptide cysteine (C), arginine (R), asparagine (N) and leucine (L) or CRNL is a potent inhibitor of tyrosinase activity with an IC50 value of 39.62 ± 6.21 µM, which is comparable to currently used tyrosinase inhibitors. Through structure-activity studies and computational modeling, we demonstrate the peptide interacts with the enzyme via electrostatic (R with E322), hydrogen bonding (N with N260) and hydrophobic (L with V248) intermolecular interactions and that a combination of these is required for potent activity. Moreover, copper chelating activity might be one of the mechanisms of tyrosinase inhibition by CRNL. Kinetic studies show that tetrapeptide is a competitive inhibitor with two-step irreversible inhibition. In addition, CRNL had no toxicity and could reduce melanin levels in the murine melanoma cell line (B16F1). Overall, CRNL is a very promising candidate for hyperpigmentation treatment.

Impact of Grinding and Sorting Particle Size on Phytochemical Yield in Dipterocarpus alatus Leaf Extract.

The main objective of this study was to investigate the impact of grinding (pretreatment) with a pin mill on the crude extract yields of Dipterocarpus alatus (Yang-Na) leaves. A factorial design in a completely randomized design was conducted to study the combinational effects of sieve sizes (1.0, 1.5, and 3.0 mm) and feed rates (1.0, 1.5, and 3.0 kg min-1), examining the interaction of parameters for grinding oven-dried Yang-Na leaves. Ethanol extraction initially evaluated the influence of Yang-Na leaf powder with diverse particle sizes. When sorting particle size, the crude extract yield increased as the particle size decreased, with 0.038-0.150 mm particles yielding the highest extraction, although yields decline when the particle size is lower than 0.038 mm. The average particle sizes, production capacity, and fineness modulus all exhibited a significant decrease as the sieve size and feeding rate were reduced, while the specific energy consumption showed an inversely proportional relationship with these parameters. Intriguingly, the crude extract yield remained independent of the average particle size. Notably, the highest yield (14.79 g kg-1) was derived from a 0.31 mm average particle size, ground with a 1.5 mm sieve and a 3 kg min-1 feeding rate. This suggests that the pretreatment, involving both grinding conditions and sorting size, has an impact on the performance of the extraction process. However, this study offers an energy-efficient alternative, advocating for using average particle sizes without prior sorting, streamlining the extraction process while maintaining substantial yields. These insights underline the crucial influence of particle size and grinding techniques, advancing our understanding of efficient herbal extraction techniques for industrial applications.

Dipterocarpol in Oleoresin of Dipterocarpus alatus Attributed to Cytotoxicity and Apoptosis-Inducing Effect.

Dipterocarpus alatus Roxb. ex G. Don is widely found in Southeast Asia. Its oleo-resin has reportedly been used in biodiesel production. Two different biodiesel production processes produce resinous byproducts, namely degumming (DG) and distillation (DT). Gas chromatography-mass spectrometry identified sesquiterpenes and triterpenes in oleo-resin, DG, and DT; and long-chain hydrocarbons in oleo-resin. High-performance liquid chromatography detected dipterocarpol as a marker compound, with the highest to lowest amounts detected in DG, DT, and oleo-resin, respectively. Oleo-resin, DG, and DT exerted more cytotoxicity than dipterocarpol, and melphalan, a chemotherapeutic drug. Oleo-resin, DG, and DT exerted cytotoxicity to a different degree in T cell leukemia (Jurkat), cervical adenocarcinoma (HeLa), and human hepatocellular carcinoma (HepG2) cells, while the highest selectivity was found in the Jurkat cells compared to the non-cancer Vero cells. Dipterocarpol exhibited the highest cytotoxicity in HepG2 cells and the lowest cytotoxicity in Jurkat cells. Oleo-resin, DG, and DT induced apoptosis in Jurkat cells. In oleo-resin, DG, and DT, dipterocarpol and other compounds may act in synergy leading to cytotoxicity and an apoptosis-inducing effect. Oleo-resin, DG, and DT could be potential sources for anticancer agents. Dipterocarpol could serve as a biomarker for follow ups on the anticancer activity of a sample from D. alatus.

1H NMR metabolic phenotyping of Dipterocarpus alatus as a novel tool for age and growth determination.

Dipterocarpus alatus belongs to Family Dipterocarpaceae that can be commonly found in Southeast Asian countries. It is a perennial plant with oval-shaped leaves and oleoresin-rich wood. It has been considered as a multipurpose plant since all parts can be practically utilized. One of the major problems for utilizing Dipterocarpus alatus is the difficulty knowing the exact age as this kind of plant is ready for multipurpose use after 20 years of age. At present, the most commonly used method for determining age of Dipterocarpus alatus is the annual ring estimation. However, this conventional method is unable to provide the high precision and accuracy of age determination due to its limitation including blurry annual rings caused by enriched oleoresin in the wood. The current study aimed to investigate the differences of 1H -NMR spectroscopy-based metabolic profiles from bark and leaf of Dipterocarpus alatus at different ages including 2, 7, 15 and 25 years. Our findings demonstrated that there is a total of 56 metabolites shared between bark and leaf. It is noticeable that bark at different ages exhibited the strongest variation and sugar or sugar derivatives that were found in higher concentrations in bark compared with those in leaf. We found that decreasing levels of certain metabolites including tagatose, 1'kestose and 2'-fucosyllactose exhibited the promising patterns. In conclusion, panel metabolites involved in the sucrose biosynthesis can precisely determine the age and growth of Dipterocarpus alatus.

Structure of the newly found green turtle egg-white ribonuclease.

Marine green turtle (Chelonia mydas) egg-white ribonuclease (GTRNase) was crystallized from 1.1 M ammonium sulfate pH 5.5 and 30% glycerol using the sitting-drop vapour-diffusion method. The structure of GTRNase has been solved at 1.60 A resolution by the molecular-replacement technique using a model based on the structure of RNase 5 (murine angiogenin) from Mus musculus (46% identity). The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 86.271, b = 34.174, c = 39.738 A, alpha = 90, beta = 102, gamma = 90 degrees . GTRNase consists of three helices and seven beta-strands and displays the alpha+beta folding topology typical of a member of the RNase A superfamily. Superposition of the C(alpha) coordinates of GTRNase and RNase A superfamily members indicates that the overall structure is highly similar to that of angiogenin or RNase 5 from M. musculus (PDB code 2bwl) and RNase A from Bos taurus (PDB code 2blz), with root-mean-square deviations of 3.9 and 2.0 A, respectively. The catalytic residues are conserved with respect to the RNase A superfamily. The three disulfide bridges observed in the reptilian enzymes are conserved in GTRNase, while one further disulfide bond is required for the structural stability of mammalian RNases. GTRNase is expressed in egg white and the fact that its sequence has the highest similarity to that of snapping turtle pancreatic RNase suggests that the GTRNase secreted from oviduct cells to form egg white is probably the product of the same gene as activated in pancreatic cells.

Enzymatic properties of newly found green turtle egg white ribonuclease.

Egg white ribonuclease was first found in green turtle eggs. The general properties were studied on substrate specificity, the optimum pH and temperature, and the effect of pH and temperature on the RNase activity. The enzyme studied was specific for poly (C) and degraded poly (U) at the lower rate and had the pH optimum at 7.0 and the optimum temperature at 40 degrees C. It was stable at alkaline range (pH 8.0-10.0) and up to 60 degrees C in pH 9.0 for 1 h, and unstable at acidic side for all temperatures. All of the properties studied showed similarity to RNase A. However, the optimum pH, broad range of optimum temperature and pH stability were different from RNase A. To evaluate the relationship of the structure and enzymatic properties, the 3D-structure of this enzyme was engineered by program MODELLER using two RNases (2BWL and 2BLZ) as starting models. The differences found in activity might be affected from the structure of micro environmental changing caused by amino acids deletion and substitution on the molecule.

The complete amino acid sequence of green turtle (Chelonia mydas) egg white ribonuclease.

Egg white ribonuclease was first found in green turtle eggs. This enzyme has been purified by CM-toyopearl cation exchange. Two isoforms (GTRNase-1 and GTRNase-2) were further separated by RP-HPLC, with the same M.W. (13 kDa) and activity. These isoforms carried one amino acid exchange of Ser and Leu at the position 37. The N-terminal sequence, ETRYEKF, was determined for the transblotted protein. Internal sequences were analyzed by protein sequencer and ESI-Q-TOF mass spectrometry for tryptic peptides (Ts). The overlapping sequences were obtained from chymotryptic peptides, CNBr fragments and ISD-MS/MS analysis. The C-terminal Ile was identified by CPase-Y. The established sequence composed of 119 residues with the molecular mass of 12,942.1 Da for GTRNase-1 and 12,967.8 Da for GTRNase-2. The comparison of sequence with known pancreatic RNases, 27 positions including catalytic residues at the position 11 and 114 were conserved. Also basic residues contributed to phosphate binding residues were conserved with the exception of Lys 66. One insertion at the position 14, and 3 deletions at the position-1, between position 64-65, and 110 and 111 were found. Two Cys residues at position 65 and 72 that form a disulfide bond in mammalian RNase were deleted and exchanged. All these difference in the sequence were similar to reptile pancreatic RNase.