Nelumbo nucifera Gaertn., an aquatic medicinal plant (Nelumbonaceae family), has a history of use in traditional medicine across various regions. Our previous study demonstrated the skin anti-aging potential of its stamen ethanolic extract by effectively inhibiting collagenase and tyrosinase enzymes. While the major constituents of this extract are well documented, there is a lack of research on the individual compounds’ abilities to inhibit skin aging enzymes. Therefore, this study aimed to evaluate the anti-aging potential of the primary flavonoids found in N. nucifera using both in silico and in vitro approaches. Our initial step involved molecular docking to identify compounds with the potential to inhibit collagenase, elastase, and tyrosinase. Among the seven flavonoids studied, kaempferol-3-O-robinobioside (Kae-3-Rob) emerged as the most promising candidate, exhibiting the highest docking scores for three skin aging-related enzymes. Subsequent enzyme-based inhibition assays confirmed that Kae-3-Rob displayed robust inhibitory activity against collagenase (58.24 ± 8.27%), elastase (26.29 ± 7.16%), and tyrosinase (69.84 ± 6.07%). Furthermore, we conducted extensive 200-ns molecular dynamics (MD) simulations, revealing the stability of the complexes formed between Kae-3-Rob and each enzyme along the MD simulation time. MM/PBSA-based binding free energy calculations indicated the considerably stronger binding affinity of Kae-3-Rob for collagenase and tyrosinase compared to elastase, which was related to the greater percentage of hydrogen bond occupations. These computational findings were consistent with the relatively high inhibitory activity of Kae-3-Rob against collagenase and tyrosinase observed in our in vitro experiment. In conclusion, the results obtained from this comprehensive study suggest that Kae-3-Rob, a key flavonoid from N. nucifera, holds significant potential as a source of bioactive compounds for anti-aging cosmeceutical and other phytopharmaceutical application.

Our previous study investigated the major flavonoids and antioxidant potential of Asian water lily (Nymphaea lotus L., family Nymphaeaceae) stamens and perianth extracts. Quercetin-3-O-rhamnoside (Que-3-Rha) and kaempferol-3-O-galactoside (Kae-3-Gal) were reported as the two most prominent flavonoids found in these extracts. Many flavonoids have been reported on the skin anti-aging effect that are useful for cosmeceutical/phytopharmaceutical application. However, Que-3-Rha and Kae-3-Gal occurring in this medicinal plant have not yet been evaluated for their ability to inhibit skin-aging enzymes. Therefore, this study aimed (1) to assess the enzyme inhibitory activity of Que-3-Rha and Kae-3-Gal, and (2) to conduct molecular modeling of these compounds against critical enzymes involved in skin aging such as collagenase, elastase, and tyrosinase. In vitro enzymatic assays demonstrated that both of the two most prominent flavonoids exhibited moderate to good inhibitory activity toward these enzymes. These experimental findings were supported by molecular docking analysis, which indicated that Que-3-Rha and Kae-3-Gal showed superior binding affinity to the target enzymes compared to the positive controls. Additionally, computational predictions suggested favorable skin permeability and no severe toxicity for both compounds. The results from molecular dynamic (MD) simulation revealed that all the complexes remained stable during the 200 ns MD simulation. Structural analyses and binding free energy calculations also supported the inhibitory potential of these two flavonoids against skin-aging enzymes. In conclusion, this study provides valuable insights into the anti-aging potential of the two major flavonoids occurring in this medicinal plant, paving the way for further development of cosmeceutical/phytopharmaceutical products targeting skin aging.

α-tocopherol is the physiologically most active form of vitamin E, with numerous biological activities, such as significant antioxidant activity, anticancer capabilities, and anti-aging properties. However, its low water solubility has limited its potential use in the food, cosmetic, and pharmaceutical industries. One possible strategy for addressing this issue is the use of a supramolecular complex with large-ring cyclodextrins (LR-CDs). In this study, the phase solubility of the CD26/α-tocopherol complex was investigated to assess the possible ratios between host and guest in the solution phase. Next, the host–guest association of the CD26/α-tocopherol complex at different ratios of 1:2, 1:4, 1:6, 2:1, 4:1, and 6:1 was studied by all-atom molecular dynamics (MD) simulations. At 1:2 ratio, two α-tocopherol units interact spontaneously with CD26, forming an inclusion complex, as supported by the experimental data. In the 2:1 ratio, a single α-tocopherol unit was encapsulated by two CD26 molecules. In comparison, increasing the number of α-tocopherol or CD26 molecules above two led to self-aggregation and consequently limited the solubility of α-tocopherol. The computational and experimental results indicate that a 1:2 ratio could be the most suitable stoichiometry to use in the CD26/α-tocopherol complex to improve α-tocopherol solubility and stability in inclusion complex formation.

• Andrographolide analogues were found to inhibit SARS-CoV-2 main protease. • The compounds 3 k, 3 l, 3 m and 3 t showed promising in vitro inhibitory activity. • Most of the candidates could bind well to the SARS-CoV-2 main protease active site.

Metal nanomaterials can enhance the efficacy of current cancer therapies. Here, we show that Ti_0.8O₂ nanosheets cause cytotoxicity in several lung cancer cells but not in normal cells. The nanosheet-treated cells showed certain apoptosis characteristics. Protein analysis further indicated the activation of the p53-dependent death mechanism. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses revealed the cellular uptake of the nanosheets and the induction of cell morphological change. The nanosheets also exhibited a substantial apoptosis effect on drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than standard drugs. Ti_0.8O₂ nanosheets induce apoptosis through a molecular mechanism involving peroxynitrite (ONOO⁻) generation. As peroxynitrite is known to be a potent inducer of S-nitrosylation, we further found that the nanosheets mediated the S-nitrosylation of p53 at C182, resulting in higher protein-protein complex stability, and this was likely to induce the surrounding residues, located in the interface region, to bind more strongly to each other. Molecular dynamics analysis revealed that S-nitrosylation stabilized the p53 dimer with a $\mathsf{\Delta }{G}_{\mathrm{bind}}^{\mathrm{residue}}$ of <−1.5 kcal/mol. These results provide novel insight on the apoptosis induction effect of the nanosheets via a molecular mechanism involving S-nitrosylation of the p53 protein, emphasizing the mechanism of action of nanomaterials for cancer therapy.

Achieving efficient enzyme catalysis under extreme pH conditions remains a major challenge in biocatalysis and synthetic biology. To address this, we present an enzyme engineering strategy that integrates rational redesign of catalytic residues with directed evolution to enable robust enzyme function at alkaline pH. The core principle involves replacing the conserved general base with an ionizable residue of higher intrinsic pK_a, shifting the proton transfer mechanism from carboxylate- to phenolate-mediated catalysis. Previously, we engineered TEM β-lactamase by substituting the universally conserved Glu166 with tyrosine (E166Y), which severely impaired activity. Directed evolution subsequently restored function, yielding the optimized variant YR5-2. Although this engineering effort originally aimed to validate a novel selection platform, the evolutionary trajectory of YR5-2 exemplifies our proposed strategy in the present study. Here, we characterize YR5-2 and its parental variants across a wide pH range. Steady-state kinetic analyses reveal a > 3-unit shift in the optimal pH for k_cat, with YR5-2 reaching 870 s^-1 at pH 10.0, a k_cat value comparable to that of the wild type at its optimal pH. Kinetic analyses of Y166E revertants, together with molecular dynamics simulations, support a mechanistic transition in which Tyr166 functions as the catalytic general base. In vivo experiments further demonstrate the utility of YR5-2 as a selectable marker by enabling recombinant protein expression in E. coli under alkaline growth conditions. This work establishes a broadly applicable framework for reprogramming enzyme catalytic mechanisms, particularly in hydrolases, to expand their operational pH range and unlock new opportunities in industrial and environmental biocatalysis.