Purpose: Inhibition of dipeptidyl peptidase-IV (DPP-IV) is an effective therapy for treating type II diabetes (T2D) that has been widely applied in clinical practice. We aimed to evaluate the DPP-IV inhibitory properties of ginger protease hydrolysate (GPH) and propose a comprehensive approach to screen and evaluate DPP-IV inhibitors. Methods: We evaluated the in vitro inhibitory properties of fish skin gelatin hydrolysates produced by five proteases, namely, neutral protease, alkaline protease, bromelain, papain, and ginger protease, toward DPP-IV. We screened the most potent DPP-IV inhibitory peptide (DIP) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with in silico analysis. Next, surface plasmon resonance (SPR) technology was innovatively introduced to explore the interactions between DPP-IV and DIP, as well as the IC₅₀. Furthermore, we performed oral administration of DIP in rats to study its in vivo absorption. Results: GPH displayed the highest degree of hydrolysis (20.37%) and DPP-IV inhibitory activity (65.18%). A total of 292 peptides from the GPH were identified using LC-MS/MS combined with de novo sequencing. Gly-Pro-Hyp-Gly-Pro-Pro-Gly-Pro-Gly-Pro (GPXGPPGPGP) was identified as the most potent DPP-IV inhibitory peptide after in silico screening (Peptide Ranker and molecular docking). Then, the in vitro study revealed that GPXGPPGPGP had a high inhibitory effect on DPP-IV (IC₅₀: 1012.3 ± 23.3 μM) and exhibited fast kinetics with rapid binding and dissociation with DPP-IV. In vivo analysis indicated that GPXGPPGPGP was not absorbed intact but partially, in the form of dipeptides and tripeptides. Conclusion: Overall, the results suggested that GPH would be a natural functional food for treating T2D and provided new ideas for searching and evaluating potential antidiabetic compounds. The obtained GPXGPPGPGP can be structurally optimized for in-depth evaluation in animal and cellular experiments.

Screening functional food ingredients (FFI) from medicinal and edible plants (MEP) has still remained a great challenge due to the complexity of MEP and its obscure function mechanisms. Herein, an integrated strategy based on sequential metabolites identification approach, network pharmacology, molecular docking, and surface plasmon resonance (SPR) analysis was proposed for quickly identifying the active constituents in MEP. First, the sequential biotransformation process of MEP, including intestinal absorption and metabolism, and hepatic metabolism, was investigated by oral gavage, and intestinal perfusion with venous sampling method. Then the blood samples were analyzed by UPLC-Q Exactive Orbitrap HRMS. Second, the network pharmacology approach was used to explore the potential targets and possible mechanisms of the in vivo metabolites of MEP. Third, molecular docking and SPR approaches were used to verify the specific interactions between protein targets and representative ingredients. The proposed integrated strategy was successfully used to explore the heptoprotective components and the underlying molecular mechanism of Paeoniae Radix Alba (PRA). A total of 44 compounds were identified in blood samples, including 17 porotypes and 27 metabolites. The associated metabolic pathways were oxidation, methylation, sulfation, and glucuronidation. After further screening, 31 bioactive candidates and 377 related targets were obtained. In addition, the bioactive components contained in PRA may have therapeutic potentials for non-alcoholic fatty liver disease (NAFLD). The above results demonstrated the proposed strategy may provide a feasible tool for screening FFI and elaborating the complex function mechanisms of MEP.

(1) Methods: An integrated strategy, including in vitro study (degree of hydrolysis (DH) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) and in vivo study (absorption after oral administration in rats), was developed to evaluate the properties of the fish skin gelatin hydrolysates prepared using different proteases (pepsin, alkaline protease, bromelain, and ginger protease). Meanwhile, in order to identify the hydrolysis site of ginger protease, the peptides in the ginger protease-degraded collagen hydrolysate (GDCH) were comprehensively characterized by liquid chromatography/tandem mass spectrometry (LC-MS) method. (2) Results: The GDCH exhibited the highest DH (20.37%) and DPPH radical scavenging activity (77.73%), and in vivo experiments showed that the GDCH was more efficiently absorbed by the gastrointestinal tract. Further oral administration experiments revealed that GDCH was not entirely degraded to free amino acids and can be partially absorbed as dipeptides and tripeptides in intact forms, including Pro-Hyp, Gly-Pro-Hyp, and X-Hyp-Gly tripeptides. LC-MS results determined the unique substrate specificity of ginger protease recognizing Pro and Hyp at the P₂ position based on the amino acids at the P₂ position from the three types of tripeptides (Gly-Pro-Y, X-Hyp-Gly, and Z-Pro-Gly) and 136 identified peptides (>4 amino acids). Interestingly, it suggested that ginger protease can also recognize Ala in the P₂ position. (3) Conclusions: This study comprehensively evaluated the properties of GDCH by combining in vitro and in vivo strategies, and is the first to identify the cleavage site of ginger protease by LC-MS technique. It provides support for the follow-up study on the commercial applications of ginger protease and bioactivities of the hydrolysate produced by ginger protease.

Rapid Resolution Liquid Chromatography coupled with Electrospray Ionization Tandem Mass Spectrometry (RRLC-ESI-MSⁿ) was used to separate and identify related substances in ampicillin capsule. The fragmentation behaviors of related substances were used to identify their chemical structures. Finally, a total of 13 related substances in ampicillin capsule were identified, including four identified components for the first time and three groups of isomers on the basis of the exact mass, fragmentation behaviors, retention time, and chemical structures in the literature. This study avoided time-consuming and complex chemosynthesis of related substances of ampicillin and the results could be useful for the quality control of ampicillin capsule to guarantee its safety in clinic. In the meantime, it provided a good example for the rapid identification of chemical structures of related substances of drugs.

Despite the increasing knowledge of biological isotope effect, comprehensive understanding of heavy isotope effect in the biological contexts has remained far less than expectation. The present study investigated the carbon isotope effect of ¹³C enriched testosterone on human cells. It was among the rare studies on carbon isotope effect of bioactive compound. Human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells were cultured in vitro and treated with testosterone and 13C enriched testosterone (¹³C/1²C:6.7%). The impacts of physiological to pharmacological concentrations (10-10-10-5mol/L) of the bioactive compound were taken into account. The cell proliferation activities were measured using MTS assay. The levels of alkaline phosphatase and osteocalcin in osteoblasts were tested. Our results established that 13C enriched testosterone exhibited different biological effects from testosterone. At the concentrations of 10-¹⁰mol/L and 10-⁵mol/L, there were significant differences in prompting cell proliferation between testosterone and ¹³C enriched testosterone. At physiological concentrations, testosterone prompted proliferations of the three kinds of cells; whereas, ¹³C enriched testosterone did not prompt the cell proliferation, and its effects were not concentration dependent. At supraphysiological concentration (10-⁵mol/L), testosterone had the trend of inhibiting cell growth; whereas, ¹³C enriched testosterone had the trend of prompting cell growth. ¹³C enriched testosterone significantly enhanced osteocalcin secretion in human osteoblasts at supraphysiological concentration. These findings challenged the common view of growth retardation effect of heavy isotope, which imply that biological isotope effects are worthy of further study. The potential applications of ¹³C enriched compound were discussed.