Previously reported peptides derived from napin of rapeseed (Brassica napus) have been shown to inhibit DPP-IV in silico. In the present study, napin extracted from rapeseed was hydrolyzed by commercial enzymes and filtered by an ultrafiltration membrane. The napin hydrolysate was then purified by a Sephadex G-15 gel-filtration column and preparative RP-HPLC. A two-enzyme-combination approach with alcalase and trypsin was the most favorable in terms of the DPP-IVinhibitory activity (IC 50 = 0.68 mg/mL) of the napin hydrolysate. Three peptides and one modified peptide (pyroglutamate mutation at the N-terminus) were identified using HPLC-triple-TOF-MS/MS. DPP-IV-inhibitory activity and the types of enzyme inhibition were also determined. Meanwhile, key residues associated with the interactions between the selected peptides and DPP-IV were investigated by molecular docking. IPQVS has key amino acid residues (Tyr547, Glu205, and Glu206) that are consistent with Diprotin A. ELHQEEPL could form a better covalent bond with Arg358 in the S3 pocket of DPP-IV.

E7974 is a synthetic analogue of the marine sponge natural product hemiasterlin. Here, we show that E7974, such as parental hemiasterlin, acts via a tubulin-based antimitotic mechanism. E7974 inhibits polymerization of purified tubulin in vitro with IC 50 values similar to those of vinblastine. In cultured human cancer cells, E7974 induces G 2 -M arrest and marked disruption of mitotic spindle formation characteristic of tubulin-targeted anticancer drugs. Extensive hypodiploid cell populations are seen in E7974-treated cells, indicating initiation of apoptosis after prolonged G 2 -M blockage. Consistent with this observation, E7974 induces caspase-3 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis. Only a short cellular exposure to E7974 is sufficient to induce maximum mitotic arrest, suggesting that E7974's antitumor effects in vivo may persist even after blood levels of the drug decrease after drug administration. Interactions of E7974 with purified tubulin were investigated using two synthetic tritiated photoaffinity analogues incorporating a benzophenone photoaffinity moiety at two different positions of the E7974 scaffold. Both analogues preferentially photolabeled α-tubulin, although minor binding to β-tubulin was also detected. E7974 thus seems to share a unique, predominantly α-tubulin-targeted mechanism with other hemiasterlin-based compounds, suggesting that, unlike many tubulin-targeted natural products and related drugs, the hemiasterlins evolved to mainly target α-tubulin, not β-tubulin subunits.

WDHHAPQLR (RAP) is an antioxidative peptide derived from rapeseed protein. Although the health benefits from RAP, due to its antioxidant activities, have been determined by chemical methods, a systematic assessment regarding the absorption, metabolism, and antioxidation processes of RAP is still lacking attention. Hence, Caco-2 cell monolayer models and animal experiments were used to evaluate the absorption and bioavailability of RAP. As expected, RAP could be absorbed by intestinal epithelial cells, and the Papp was 0.82 ± 0.19 × 10 cm/s. Three main fragments, RAP, DHHAPQLR, and WDHHAP were transported by the paracellular pathway, and QLR was transported by PepT1. An important modified product of RAP (EGDHHAPQLR) was found to contribute to the elimination of intracellular reactive oxygen species. The absolute bioavailability of RAP was 3.56%, and three degradation products of RAP were also detected in rat serum. More importantly, RAP exerts its antioxidant activity by inhibiting the apoptosis of oxidative stress cells. RAP could downregulate the expression of Bax and caspase-3 and upregulate the expression of Bcl-2 in HO-induced HUVECs (human umbilical vein endothelial cells). In general, using in vitro and in vivo experimental models, the in vivo absorption and transformation processes of RAP and its antioxidative molecular mechanisms by inhibiting apoptosis of cells were revealed.

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Polyphenol extracts derived from gastrointestinal digestates of buckwheat (Fagopyrum Mill) were studied for their intestinal transport and lipid-lowering effects in Caco-2/HepG2 coculture models. The relative amounts of all phenolic compounds throughout the digestion and intestinal absorption process were determined by UHPLC-Q-Orbitrap mass spectrometry. The digestible and easily transported phenolic compounds in buckwheat extract were identified. Herein, four main phenolic compounds and their metabolites were found on both the apical and basolateral sides of the Caco-2 cell transwell model. The transepithelial transport rates in the Caco-2 cell monolayer were scoparone (0.97) > hydroxycinnamic acid (0.40) > rutin (0.23) > quercetin (0.20). The main metabolism of hydroxycinnamic acid, quercetin, and scoparone in transepithelial transport was found to be methylation. Furthermore, results indicated that triglyceride, low-density lipoprotein cholesterol, total cholesterol, aspartate aminotransferase, and alanine aminotransferase levels in HepG2 cells on the basolateral side of coculture models can be suppressed by 53.64, 23.44, 36.49, 27.98, and 77.42% compared to the oleic acid-induced group (p < 0.05). In addition, the mRNA expression of Fabp4 relative to the control was found to be significantly upregulated (85.82 ± 10.64 to 355.18 ± 65.83%) by the easily transported buckwheat polyphenol components in HepG2 cells (p < 0.01).

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