The ability of casein phosphopeptides (CPPs) to bind and transport minerals has been previously studied. However, the single bioactive peptides responsible for the effects of CPPs have not been identified. This study was to purify calcium-binding peptides from CPPs and to determine their effects on calcium and magnesium uptake by Caco-2 cell monolayers. Five monomer peptides designated P1 to P5 were isolated and the amino acid sequences were determined using LC-MS/MS. Compared with the CPP-free control, all five monomeric peptides exhibited significant enhancing effects on the uptake of calcium and magnesium (P < 0.05). Interestingly, when calcium and magnesium were presented simultaneously with P5, magnesium was taken up with priority over calcium in the Caco-2 cell monolayers. For example, at 180 min, the amount of transferred magnesium and calcium was 78.4 ± 0.95 μg/well and 2.56 ± 0.64 μg/well, respectively, showing a more than 30-fold difference in the amount of transport caused by P5. These results provide novel insight into the mineral transport activity of phosphopeptides obtained from casein.Keywords: Caco-2 cell monolayers; calcium transport; casein phosphopeptides; identification; magnesium transport;

The tender leaves of Rubus corchorifolius L. f. have been consumed as tea for drinking in China since ancient times. In this study, a novel ent-kaurane diterpenoid was isolated and identified from R. corchorifolius L. f. leaves as ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal (DEK). DEK suppressed the growth of HCT116 human colon cancer cells with an IC50 value of 40 ± 0.21 μM, while it did not cause significant growth inhibition on CCD-18Co human colonic myofibroblasts at up to100 μM. Moreover, DEK induced extensive apoptosis and S phase cell cycle arrest in the colon cancer cells. Accordingly, DEK caused profound effects on multiple signaling proteins associated with cell proliferation, cell death, and inflammation. DEK significantly upregulated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, cleaved caspase-9, cleaved PARP, p53, Bax, and tumor suppressor p21Cip1/Waf1, downregulated the levels of cell cycle regulating proteins such as cyclinD1, CDK2, and CDK4 and carcinogenic proteins such as EGFR and COX-2, and suppressed the activation of Akt. Overall, our results provide a basis for using DEK as a potential chemopreventive agent against colon carcinogenesis.Keywords: apoptosis; cell cycle; colon cancer; ent-kaur-2-one-16β,17-dihydroxy-acetone-ketal; Rubus corchorifolius L.f.;

The effect of dietary magnesium (Mg) or caseinphosphopeptides (CPPs) on bone metabolism has been reported. However, few studies have investigated the effects of simultaneous supplementation of Mg and CPPs. Sixty-three 3-week-old Sprague-Dawley male rats were divided into seven groups and fed a specified diet for 45 days. Body characteristics, bone physicochemical indicators, and bone metabolism indicators relative to bone metabolism were analyzed. We found that, first, a dietary Mg deficiency resulted in increased bone formation and decreased bone resorption. Second, dietary Mg or CPP supplementation promoted bone formation and prevented bone resorption. Third, dietary Mg supplementation with CPPs also functioned to enhance bone formation and prevent bone resorption. There were synergistic effects on femur length, serum parathyroid hormone level and urinary deoxypyridinoline of the HS-Mg-CPP group (0.2% Mg, 0.1% CPPs). The increase in the femur length of the HS-Mg-CPP group compared with the control group was 6% which was much higher than that of HS-Mg (1%) or CPPs (5%). The induction in serum parathyroid hormone content in the HS-Mg-CPP group was 33% compared with the control group which was higher than that of the induction of the HS-Mg (19%) or CPP (23%) group. The induction in the deoxypyridinoline content of the HS-Mg-CPP (43%) group compared with the control group was remarkably higher than that of HS-Mg (8%) or CPPs (16%). Overall our results demonstrated that high doses of Mg (0.2%) and CPPs (0.1%) in combination produced synergistic effects on femur length, serum parathyroid hormone level and urinary deoxypyridinoline in rats, which is important for a better understanding of the effect of Mg and CPPs on bone metabolism.

•Chenpi extract lowers lipid accumulation in differentiating adipocytes.•Chenpi extract does not significantly affect mitotic clonal expansion.•Chenpi extract does not significantly affect preadipocyte proliferation.•Chenpi extract lowers expression of adipogenic regulators and lipogenic genes.•5-OH PMFs content contributes to the anti-lipogenic activity of chenpi extract.Aged citrus peel (chenpi) is made from the dry peel of the fruit of Citrus reticulate Blanco after aging process. It has long been used as a food ingredient, a dietary supplement, and for medicinal purpose. Compared to fresh citrus peel extract, chenpi extract contains more 5-demethylated polymethoxyflavones (5-OH PMFs). To investigate a potential direct role of chenpi extract on adipose tissue, we examined the effect of chenpi extract on differentiating 3T3-L1 cells. We showed that two types of chenpi extract varying in 5-OH PMF contents consistently reduces intracellular lipid accumulation without significantly affecting cell viability and proliferation. The reduction in lipid accumulation is correlated with AMP-activated protein kinase (AMPK) activation and down-regulation of adipogenic transcription factors as well as lipogenic genes. The results indicate that chenpi extract may directly affect lipogenesis in adipose tissue and the content of 5-OH PMFs likely affects the anti-lipogenic activity of chenpi extract.

The leaves of Rubus corchorifolius L. f. have been consumed as a herbal tea for a long time. In this study, two novel (1 and 5) and four known (2, 3, 4 and 6) terpenoids were isolated from the leaves of Rubus corchorifolius L. f. Structural analysis was performed using various spectroscopic methods (1H NMR, 13C NMR and MS) to identify the following six compounds: (16α)-16,17,18-trihydroxy-ent-kauran-18-O-β-D-glucoside (1), ent-16β,17-dialkyl-3-oxygen-kaurane (2), ent-kaurane-3α,16β,17-triol (3), ent-kaurane(5R,8S,9R,10R,13R,16R)-2-one-16α,17-diol (4), (16R)-16β,17,19-trihydroxy-ent-kaur-3-one (5) and ent-16α,17-dihydroxy-kauran-19-oic-acid (6). These compounds showed different inhibitory effects on various human cancer cells. Compounds 3 and 6 exhibited stronger inhibitory effects on human colon cancer HCT116 cells than the other 4 compounds. Flow cytometry analysis demonstrated that both compounds 3 and 6 caused cell cycle arrest at the G0/G1 phase and induced cellular apoptosis in HCT116 cells. Compounds 3 and 6 modulated the expression levels of key signaling proteins closely related to cell proliferation and apoptosis, i.e., increasing the levels of poly(ADP-ribose) polymerase, p53, and p27, and decreasing the levels of EGFR, cyclin D1, CDK2 and CDK4. Overall, our findings provided insight into the anticancer components of Rubus corchorifolius L. f. leaves, which could facilitate their utilization as functional food ingredients.

Kefir is a traditional fermented milk beverage used throughout the world for centuries. A cell-penetrating peptide, F3, was isolated from kefir by Sephadex G-50 gel filtration, DEAE-52 ion exchange, and reverse-phase high-performance liquid chromatography. F3 was determined to be a low molecular weight peptide containing one leucine and one tyrosine with two phosphate radicals. This peptide displayed antimicrobial activity across a broad spectrum of organisms including several Gram-positive and Gram-negative bacteria as well as fungi, with minimal inhibitory concentration (MIC) values ranging from 125 to 500 μg/mL. Cellular penetration and accumulation of F3 were determined by confocal laser scanning microscopy. The peptide was able to penetrate the cellular membrane of Escherichia coli and Staphylococcus aureus. Changes in cell morphology were observed by scanning electron microscopy (SEM). The results indicate that peptide F3 may be a good candidate for use as an effective biological preservative in agriculture and the food industry.

Tannases produced by filamentous fungi are in a family of important hydrolases of gallotannins and have broad industry applications. But until now, the 3-D structures of fungi tannases have not been reported. The protein sequence deduced from the cDNA sequence obtained using RT-PCR amplification was identified as tannase through sequence alignment and phylogenetic analysis. Structure models based on the tannase sequence were collected using I-TASSER, and the model with the best match to the surface charge density-pH titration profile was selected as the final structure for tannase from Aspergillusniger N5-5. This work provides an effective method for protein structure research. The structure constructed in this work should be very important to understand the enzyme bioactivities and further developments of fungi tannases.The protein sequence deduced from the cloned tannase gene sequence from Aspergillus niger N5-5 was identified as tannase through sequence alignment and phylogenetic analysis. Then, the enzyme 3-D structure was predicted using I-TASSER and experimentally validated.

•The antimicrobial substances were produced by probiotic Lactobacillus paracasei.•The optimized conditions increased the antimicrobial activity.•The antimicrobial substances exhibited a broad antimicrobial spectrum.•The antimicrobial substances were resistant to high temperature and insensitive to digestive enzymes.•Three active components were isolated from the antimicrobial substances.There is an increasing demand from consumers for natural antimicrobial substances that can be used for food preservation. This study is focused on establishing the conditions to enhance the production of antimicrobial substances by the probiotic Lactobacillus paracasei Subsp. Tolerans FX-6 from Tibetan kefir. The culture conditions were optimized by using response surface methodology, and remarkably the optimized conditions increased the antimicrobial activity by more than 80%. Further characterization revealed that the antimicrobial substances exhibited a broad spectrum of antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria and fungi. Moreover, the antimicrobial substances were highly tolerant to heat and enzyme treatment. Excitingly, the antimicrobial substances were fractionated by HPLC and the sub-fractions generated showed stronger antimicrobial activity than nisin, a highly effective bacteriocin currently used as a food preservative. Overall, these results demonstrate a novel approach to produce effective antimicrobial substances for use as natural preservatives in food.

Antimicrobial peptides have received increasing attention in the agricultural and food industries due to their potential to control pathogens. However, to facilitate the development of novel peptide-based antimicrobial agents, details regarding the molecular mechanisms of these peptides need to be elucidated. The aim of this study was to investigate the antimicrobial mechanism of peptide F1, a bacteriocin found in Tibetan kefir, against Escherichia coli at protein levels using iTRAQ-based quantitative proteomic analysis. In response to treatment with peptide F1, 31 of the 280 identified proteins in E. coli showed alterations in their expression, including 10 down-regulated proteins and 21 up-regulated proteins. These 31 proteins all possess different molecular functions and are involved in different molecular pathways, as is evident in referencing the Kyoto Encyclopedia of Genes and Genomes pathways. Specifically, pathways that were significantly altered in E. coli in response to peptide F1 treatment include the tricarboxylic acid cycle, oxidative phosphorylation, glycerophospholipid metabolism, and the cell cycle–caulobacter pathways, which was also associated with inhibition of the cell growth, induction of morphological changes, and cell death. The results provide novel insights into the molecular mechanisms of antimicrobial peptides.

•Nine polyphenols were isolated from the ethyl acetate extract of Eucalyptus leaves.•These compounds showed free radical scavenging ability in DPPH and ABTS+ assays.•Ethyl acetate extract showed high cellular antioxidant activity.•PGG exhibited the highest antioxidant activity among five hydrolysable tannins.An activity-guided isolation process was performed to identify the antioxidant constituents of Eucalyptus leaves (Eucalyptus grandis × Eucalyptus urophylla GL9). The ethyl acetate extract showed the highest activity in scavenging DPPH radical among five different fractions extracted from 70% aqueous ethanol extract of Eucalyptus leaves. Nine known compounds possessing radical scavenging activity, which included gallic acid (1), quercetin-3-O-β-d-glucuronide (Q-3-G-A) (2), ethyl gallate (3), tellimagrandin II(4), chlorogenic acid (5), 3-O-galloyl-4,6-O-[(S)-hexahydroxydiphenoyl]-d-glucose (gemin D) (6), pedunculagin (7), tellimagrandin I (8), and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) (9) were isolated and identified from the ethyl acetate extract. Our results indicated that ethyl acetate extract and hydrolysable tannins among nine compounds exhibited great antioxidant activity in both chemical-based (e.g. DPPH and ABTS assays) and cellular-based (i.e., Hep G2) antioxidant assays, and PGG showed the highest cellular antioxidant activity. This study suggested that ethyl acetate extract and the hydrolysable tannins in Eucalyptus leaves would be promising natural antioxidants in pharmaceutical, feed and food supplement industries.

On the basis of previous single-factor experiments, extraction parameters of soy sauce residue (SSR) oil extracted using a self-developed continuous phase transition extraction method at low temperature was optimized using the response surface methodology. The established optimal conditions for maximum oil yield were n-butane solvent, 0.5 MPa extraction pressure, 45 °C temperature, 62 min extraction time, and 45 mesh raw material granularity. Under these conditions, the actual yield was 28.43% ± 0.17%, which is relatively close to the predicted yield. Meanwhile, isoflavone was extracted from defatted SSR using the same method, but the parameters and solvent used were altered. The new solvent was 95% (v/v) ethanol, and extraction was performed under 1.0 MPa at 60 °C for 90 min. The extracted isoflavones, with 0.18% ± 0.012% yield, mainly comprised daidzein and genistein, two kinds of aglycones. The novel continuous phase transition extraction under low temperature could provide favorable conditions for the extraction of nonpolar or strongly polar substances. The oil physicochemical properties and fatty acids compositions were analyzed. Results showed that the main drawback of the crude oil was the excess of acid value (AV, 63.9 ± 0.1 mg KOH/g) and peroxide value (POV, 9.05 ± 0.3 mmol/kg), compared with that of normal soybean oil. However, through molecular distillation, AV and POV dropped to 1.78 ± 0.12 mg KOH/g and 5.9 ± 0.08 mmol/kg, respectively. This refined oil may be used as feedstuff oil.

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.

A novel continuous subcritical n-butane extraction technique for Camellia seed oil was explored. The fatty acid composition, physicochemical properties, and benzo[a]pyrene content of Camellia seed oil extracted using this subcritical technique were analyzed. Orthogonal experiment design (L9(34)) was adopted to optimize extraction conditions. At a temperature of 45 °C, a pressure of 0.5 MPa, a time of 50 min and a bulk density of 0.7 kg/L, an extraction yield of 99.12 ± 0.20 % was obtained. The major components of Camellia seed oil are oleic acid (73.12 ± 0.40 %), palmitic acid (10.38 ± 0.05 %), and linoleic acid (9.15 ± 0.03 %). Unsaturated fatty acids represent 83.78 ± 0.03 % of the total fatty acids present. Eight physicochemical indexes were assayed, namely, iodine value (83.00 ± 0.21 g I/100 g), saponification value (154.81 ± 2.00 mg KOH/g), freezing-point (−8.00 ± 0.10 °C), unsaponifiable matter (5.00 ± 0.40 g/kg), smoke point (215.00 ± 1.00 °C), acid value (1.24 ± 0.03 mg KOH/g), refrigeration test (transparent, at 0 °C for 5.5 h), and refractive index (1.46 ± 0.06, at 25 °C). Benzo[a]pyrene was not detected in Camellia seed oil extracted by continuous subcritical n-butane extraction. In comparison, the benzo[a]pyrene levels of crude Camellia seed oil extracted by hot press extraction and refined Camellia seed oil were measured at 26.55 ± 0.70 and 5.69 ± 0.04 μg/kg respectively.

Carnosic acid is the primary rosemary-derived phenolic diterpenes with greatest antioxidant activity, but it is rather unstable in methanol solution. Understanding the degradation pathway of carnosic acid and the interaction between these phenolic compounds is important if they are to be utilized to a greatest effect. In this study, the HPLC analysis of an aged methanolic solution of carnosic acid revealed that carnosic acid was degraded, and several compounds were produced in the solution. From the time-course and quantitative analyses of the formation of the products and their structural analysis, a novel degradation pathway of carnosic acid in methanol solution is proposed. Carnosic acid can firstly decompose into carnosic acid quinone in methanol, which was the first time to be confirmed the existence of carnosic acid quinone. Carnosic acid quinone is the quinone intermediate of carnosic acid that can be reduced into carnosic acid or can be further converted into carnosol. Carnosol can further decompose to form epirosmanol, rosmanol, 7-methoxyrosmanol, and 7-methoxy-epirosmanol. The latter three compounds were identified as the final degradation products of carnosic acid in methanol because they appeared rather stable in the study.

Sustaining the release of therapeutic nanoparticles in a cell-, tissue-, or disease-specific manner is a potentially powerful technology. A new drug carrier-dialdehyde starch nanoparticle (DASNP) that can sustain the loading and release of 5-fluorouracil (5-Fu) antitumor drug is reported in this study. IR spectrophotometer and 1H NMR confirmed the formation of aldehyde groups, and scan electron microscope determinations showed that the dialdehyde starch nanoparticles obtained had an average diameter of 90 nm. 5-Fu, the model drug, was conjugated into nanoparticles by aldehyde groups. These 5-Fu-binding nanoparticles significantly enhanced breast cancer cell (MCF-7) inhibition in vitro compared with free 5-Fu. After subcutaneous 0 injection in the breast tumor-loaded rats, 5-Fu-DASNP exhibited remarkable tumor-inhibitory efficacy determined by measuring tumor weight in vivo. The tumor inhibition of 5-Fu-DASNP was 61%±6%, whereas that of free 5-Fu was only 42%±4%. Bcl-2/Bax immunohistochemistry studies indicated that 5-Fu-DASNP remarkably induced tumor tissue necrosis. These results demonstrated that the DASNP prepared in this work is a potentially effective drug carrier.