Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Preparation, characterization, and osteogenic activity mechanism of casein phosphopeptide-calcium chelate.

Casein phosphopeptides (CPPs) are good at calcium-binding and intestinal calcium absorption, but there are few studies on the osteogenic activity of CPPs. In this study, the preparation of casein phosphopeptide calcium chelate (CPP-Ca) was optimized on the basis of previous studies, and its peptide-calcium chelating activity was characterized. Subsequently, the effects of CPP-Ca on the proliferation, differentiation, and mineralization of MC3T3-E1 cells were studied, and the differentiation mechanism of CPP-Ca on MC3T3-E1 cells was further elucidated by RNA sequencing (RNA-seq). The results showed that the calcium chelation rate of CPPs was 23.37%, and the calcium content of CPP-Ca reached 2.64 × 105 mg/kg. The test results of Ultraviolet-Visible absorption spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) indicated that carboxyl oxygen and amino nitrogen atoms of CPPs might be chelated with calcium during the chelation. Compared with the control group, the proliferation of MC3T3-E1 cells treated with 250 µg/mL of CPP-Ca increased by 21.65%, 26.43%, and 28.43% at 24, 48, and 72 h, respectively, and the alkaline phosphatase (ALP) activity and mineralized calcium nodules of MC3T3-E1 cells were notably increased by 55% and 72%. RNA-seq results showed that 321 differentially expressed genes (DEGs) were found in MC3T3-E1 cells treated with CPP-Ca, including 121 upregulated and 200 downregulated genes. Gene ontology (GO) revealed that the DEGs mainly played important roles in the regulation of cellular components. The enrichment of the Kyoto Encyclopedia of Genes and Genomes Database (KEGG) pathway indicated that the AMPK, PI3K-Akt, MAPK, and Wnt signaling pathways were involved in the differentiation of MC3T3-E1 cells. The results of a quantitative real-time PCR (qRT-PCR) showed that compared with the blank control group, the mRNA expressions of Apolipoprotein D (APOD), Osteoglycin (OGN), and Insulin-like growth factor (IGF1) were significantly increased by 2.6, 2.0 and 3.0 times, respectively, while the mRNA levels of NOTUM, WIF1, and LRP4 notably decreased to 2.3, 2.1, and 4.2 times, respectively, which were consistent both in GO functional and KEGG enrichment pathway analysis. This study provided a theoretical basis for CPP-Ca as a nutritional additive in the treatment and prevention of osteoporosis.

The purification, identification and bioactivity study of a novel calcium-binding peptide from casein hydrolysate.

In this study, a novel calcium-binding peptide from casein hydrolysate was purified using reversed-phase high performance liquid chromatography and sequenced by high-performance liquid chromatography-mass spectrometry (MS)/MS. The amino acid sequence of the calcium-binding peptide was identified as VLPVPQK (N- to C-terminal, MW = 779.4960 Da). The calcium binding characteristics of VLPVPQK were further investigated using UV absorption spectroscopy, zeta potential and isothermal titration calorimetry (ITC). The results showed that VLPVPQK has a strong calcium binding activity (129.46 mg g-1), 312% higher than that of 3-hour enzymatic hydrolysates. VLPVPQK could chelate calcium with a 1 : 3 stoichiometry, causing a decrease in the positive charge of the peptide-Ca2+ complex. Furthermore, VLPVPQK could effectively enhance calcium transport and absorption in a concentration-dependent manner in Caco-2 cell monolayers, suggesting that VLPVPQK has the potential to be developed as a nutraceutical additive.

Isolation and identification of iron-chelating peptides from casein hydrolysates.

Iron deficiency is a common nutritional disorder worldwide. Peptides derived from protein hydrolysates have recently attracted interest as novel iron chelators due to their superiority in terms of increasing solubility, bioavailability, absorption and stability. The aim of this study was to isolate and identify iron-chelating peptides from casein hydrolysates. Casein was hydrolyzed (trypsin, 3 h) and subsequently isolated using ultrafiltration and RP-HPLC. Four iron-chelating casein hydrolysate peptides, named CHP-1, CHP-2, CHP-3 and CHP-4, were identified by LC-MS/MS, and their amino acid sequences were Glu-Asp-Val-Pro-Ser-Glu-Arg (EDVPSER), His-Lys-Glu-Met-Pro-Phe-Pro-Lys (HKEMPFPK), Asn-Met-Ala-Ile-Asn-Pro-Ser-Lys (NMAINPSK) and Ala-Val-Pro-Tyr-Pro-Gln-Arg (AVPYPQR), with molecular weights of 830.6120 Da, 1012.5280 Da, 873.4440 Da and 829.4570 Da, respectively. The artificially synthesized peptides of CHP-1, CHP-2, CHP-3 and CHP-4 were verified, and their iron-chelating rates were 11.14%, 8.02%, 7.57% and 59.76%, respectively. These results suggested that the isolated iron-chelating peptides might serve as potential iron supplements and be used as food additives and functional foods.

Anti-fatigue and anti-oxidant activities of oyster (Ostrea rivularis) hydrolysate prepared by compound protease.

Oyster, which is rich in protein and widely used as a marine traditional Chinese medicine, was believed to have good curative effects in health care and on chronic diseases. This study was designed to evaluate the anti-fatigue and anti-oxidant effects of oyster hydrolysate. Oyster meat (OM) was hydrolyzed with a complex protease, and oyster hydrolysate (OH) was separated by a 6 kDa ultrafiltration membrane into two fractions, OH-I (<6 kDa) and OH-II (≥6 kDa). The anti-fatigue effects of OM, OH, OH-I and OH-II groups were first investigated, and then the antioxidant activities of OH-I and OH-II were further analyzed. Anti-fatigue experimental results showed that OH-I displayed the strongest activity among the four groups. Compared to the control group, OH-I significantly prolonged swimming time (67.79%), increased the content of muscle glycogen (45.65%) and liver glycogen (49.01%), and reduced the content of blood urea nitrogen (BUN) (18.44%) (P < 0.05). Meanwhile, OH-I showed excellent chemical and cellular antioxidant activities, especially when the concentration increased; its antioxidant activity was significantly better than that of OH-II (P < 0.05). Results of an amino acid analysis showed that OH-I was rich in branched-chain amino acids (10.84 g per 100 g), Glu (8.63 g per 100 g), Tau (1.68 g per 100 g), Asp (5.02 g per 100 g) and Arg (3.61 g per 100 g), which were expected to contribute to its antioxidant and anti-fatigue activities.

Mechanism of longevity extension of Caenorhabditis elegans induced by pentagalloyl glucose isolated from eucalyptus leaves.

The multicellular model organism Caenorhabditis elegans (C. elegans) was used to identify the anti-aging effect of pentagalloyl glucose (PGG) isolated from Eucalyptus leaves at four different concentrations. For 160 µM PGG, the median lifespan of C. elegans was found to increase by 18%, and the thermal stress resistance was also increased. The anti-aging effect of PGG did not cause side effects on the physiological functions including the reproduction, pharyngeal pumping rate, age pigments accumulation, and locomotion ability. The life extension induced by PGG was found to rely on genes daf-16, age-1, eat-2, sir-2.1, and isp-1 but did not rely on genes mev-1 and clk-1. These findings suggested that the insulin/IGF-1 signaling pathway, dietary restriction, Sir-2.1 signaling, and mitochondrial electron transport chain became partly involved with the mechanism of lifespan extension mediated by PGG. Our results provided an insight into the mechanism of longevity extension mediated by PGG in C. elegans, which might be developed into a new generation of multitarget drug to prolong lifespan.

[Studies on the chemical components and antibacterial activity of essential oil from the leaves of Rubus corchorifolius].

OBJECTIVE: To analyze the chemical components of essential oil from the leaves of Rubus corchorifolius. METHODS: The essential oil was extracted by steam distillation, and then an analysis was conducted by applying GC-MS and Xcalibur Software. K-B filter paper method was used for their bacteriostasish. RESULTS: 44 compounds were identified, accounting for 88.52% of the total essential oil. The volatile oil concentration of 50% had a good bactericidal effect and the concentration of 0.5% still had excellent antibacterial effect. CONCLUSION: It provides the foundation for reasonable utilization and development of the leaves of Rubus corchorifolius.

Bioconjugated nanoparticle for DNA protection from ultrasound damage.

To research whether poly-L-lysine-starch nanoparticle (PLL-StNP) could protect DNA from ultrasound damage or not, a series experiments were carried out: plasmid DNA-PLL-StNP complexes were treated with ultrasound for diverse times; the electrophoresis result proved that DNA bound to the complexes all the same. To investigate whether the conjugated DNA was completely protected or not, cDNA fragments bound to PLL-StNP were treated with ultrasound, and matched molecular beacons (MBs) were added. The cDNA-MB-PLL-StNP complexes exhibited dramatically increasing fluorescence, and had the same intensity as that of those MBs that were hybridized with free cDNA fragments. After being treated by ultrasound, the pIRGFP plasmid DNA-PLL-StNP complexes were transferred into COS-7 cells mediated by ultrasound. Green fluorescence protein expressed in most of the cells. Those results indicated that PLL-StNP could completely protect DNA from ultrasound damage. Furthermore, the DNA kept the same function as untreated one.