The cytotoxic effect of Bowman–Birk isoinhibitors,IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman–Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health‐promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non‐malignant colonic fibroblast CCD‐18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9±2.3 and 48.3±3.5 μM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose‐dependent manner, with cells becoming blocked in the G0–G1 phase. Chemically inactive soybean BBI had a weak but non‐significant effect on the proliferation of HT29 cells. The anti‐proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin‐ and chymotrypsin‐like proteases involved in carcinogenesis should be considered as potential targets of BBI‐like proteins.     

Cytotoxic impact of phenolics from Lamiaceae species on human breast cancer cells

The aim of this study was to evaluate the cytotoxicity of dried aqueous extracts from Thymus serpyllum (ExTs), Thymus vulgaris (ExTv), Majorana hortensis (ExMh), and Mentha piperita (ExMp), and the phenolic compounds caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), luteolin-7-O-glucuronide (Lgr), luteolin-7-O-rutinoside (Lr), eriodictiol-7-O-rutinoside (Er), and arbutin (Ab), on two human breast cancer cell lines: Adriamycin-resistant MCF-7/Adr and wild-type MCF-7/wt. In the MTT assay, ExMh showed the highest cytotoxicity, especially against MCF-7/Adr, whereas ExMp was the least toxic; particularly against MCF-7/wt cells. RA and LA exhibited the strongest cytotoxicity against both MCF-7 cell lines, over 2-fold greater than CA and Lgr, around 3-fold greater than Er, and around 4- to 7-fold in comparison with Lr and Ab. Except for Lr and Ab, all other phytochemicals were more toxic against MCF-7/wt, and all extracts exhibited higher toxicity against MCF-7/Adr. It might be concluded that the tested phenolics exhibited more beneficial properties when they were applied in the form of extracts comprising their mixtures.     

The potential of sphingomyelin as a chemopreventive agent in AOM-induced colon cancer model: wild-type and p53+/- mice

A protective effect of sphingolipids on colorectal cancer (CRC) has been reported in certain mouse strains. It is unknown if sphingolipids are protective in a p53 deficiency mouse model of CRC. This study investigated the effect of sphingomyelin (SM) on intestinal sphingomyelinase (SMase) activity, colonic epithelial biology and azoxymethane (AOM)-induced CRC. Groups of wild-type (C57BL/6J) and p53+/- mice were fed 0.1% SM diet for 4 wk, administered a single AOM injection and then killed 6 h later to measure apoptosis and proliferation. Separately, both mouse types were fed 0.05% SM diet, administered three AOM injections and killed 33-38 wk later to measure tumour formation. SM significantly increased SMase activity and reduced proliferation (p < 0.05) in wild-type and p53+/- mice. SM did not regulate baseline apoptosis, apoptotic response to AOM or apoptosis in tumours, nor did it restore defective apoptosis in p53+/- mice. There was a nonsignificant trend to reduced tumour incidence with SM in wild-type (p = 0.15) and p53+/- (p = 0.12) mice. In conclusion, while increasing intestinal SMase activity and suppressing proliferation, SM did not promote any form of apoptosis and failed to achieve significant protection in these mice. Further investigation to understand the variable effect of SM in preventing CRC is warranted.     

Constituents isolated from Glehnia littoralis suppress proliferations of human cancer cells and MMP expression in HT1080 cells

Crude extract and solvent-partitioned fraction of Glehnia littoralis were found to possess different anti-proliferative effects against AGS, HT1080 and U937 human cancer cells. The crude extracts and solvent fractions dose-dependently inhibited cell proliferation. Especially, n-hexane and 85% aqueous MeOH fractions exhibited comparatively higher anti-proliferative effects and reduced expressions of Bcl-2, COX-2 and iNOS genes. Systematic separation of all solvent fractions by chromatographic methods led to the isolation of three glucopyranosides, four furanocoumarins and two polyacetylenic alcohols. All the nine compounds were evaluated for their inhibitory effects against both proliferation of human cancer cells and expressions of MMP-2 and -9 in HT1080 cells. Two polyacetylenic alcohols exerted the highest inhibitory activity against both human cancer cell lines, and MMP-2 and -9. These results suggest that G. littoralis may possibly be used as a valuable chemopreventive agent or food supplement for reducing cancer risk.     

Cell proliferation inhibitory and apoptosis-inducing properties of anacardic acid and lunasin in human breast cancer MDA-MB-231 cells

The combination of two or more chemopreventive agents is currently being used to achieve greater inhibitory effects on breast cancer cells. Anacardic acid and lunasin are two plant-derived compounds that have been associated with anti-carcinogenic properties. These compounds show inhibitory effects on cell proliferation and inducing properties of apoptosis in human breast cancer MDA-MB-231 cell line. Both lunasin and anacardic acid exert their effects through the modulation of expression of several genes involved in cell cycle, apoptosis and signal transduction. Their combination arrests the cell cycle in S-phase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signalling gene expression, whose up-regulation has been reported as responsible for breast cancer cells growth and resistance to apoptosis. Our results introduce these two compounds as a promising strategy to prevent/treat breast cancer.     

Apoptosis-mediated proliferation inhibition of human colon cancer cells by volatile principles of Citrus aurantifolia

Fruits of Citrus aurantifolia were subjected to hydro-distillation using Clevenger type apparatus to obtain volatile oil. Chemical composition of volatile oil was analysed by GC–MS. Twenty-two compounds representing more than 89.5% of the volatile oil were identified. d-limonene (30.13%) and d-dihydrocarvone (30.47%) were found to be the major compounds in the lime volatile oil. This oil showed 78% inhibition of human colon cancer cells (SW-480) with 100 μg/ml concentration at 48 h. Lime volatile oil showed DNA fragmentation and induction of caspase-3 up to 1.8 and 2- folds after 24 h and 48 h, respectively, which may be due to the involvement of apoptosis. Analysis of apoptosis-related protein expression further confirmed apoptosis induction by lime volatile oil. The above results suggested that lime volatile oil has potential benefits in colon cancer prevention. This is the first report, showing the possible mechanism of antiproliferative effect of lime volatile oil for the prevention of colon cancer in cell culture models.     

Protein isolates from two Mediterranean legumes: Lathyrus clymenum and Lathyrus annuus. Chemical composition,functional properties and protein characterisation

Protein isolates were analysed from two Mediterranean legumes, Lathyrus clymenum and L. annuus. Protein isolates were prepared by alkaline extraction, including sodium sulphite and acid precipitation of Lathyrus proteins at their isoelectric point (pH 4.5). The percentage of proteins recovered from L. annuus and L. clymenum flours during the preparation of the protein isolates was around 60%. Chemical composition, nutritional parameters, main functional properties and protein composition of Lathyrus protein isolates were studied. L. annuus and L. clymenum protein isolates contained 81.07% and 82.4% of proteins, respectively, and they have a balanced content of essential amino acids, except for sulphur amino acids, with respect to the FAO pattern. The in vitro protein digestibility increased in the protein isolates to 93% and 95% in L. annuus and L. clymenum, respectively. Functional properties were similar to those observed in other legumes protein isolates. These results confirm the interest of local crops as sources of high value protein products obtained after convenient protein extraction procedures and the removal of antinutritional components. These high added value protein isolates are of interest for the food industry and for the revalorisation of L. annuus and L. clymenum favouring the bioconservation of Lathyrus.     

Chemical composition,functional properties,and bioactivities of rapeseed protein isolates

In order to utilize rapeseed protein from oil industry waste for food applications, rapeseeds were pretreated to remove the oil using hexane. Two protein isolates were prepared, one by precipitation at controlled pH and the other by ultrafiltration. The precipitated and ultrafiltered protein isolates, respectively, contained 70.8% and 98.7% protein. The ultrafiltered protein isolate had a better emulsification capacity than had whole egg. The ultrafiltered protein isolate had a protein solubility of 52.5–97.2% in the range pH 3–9, whilst the maximum protein solubility of the precipitated protein isolate was 26.4% in the pH range 7–9. There were no significant differences between the precipitated and ultrafiltered protein isolates regarding their angiotensin converting enzyme inhibition are their bile acid-binding capacity. Their bile acid-binding capacity and angiotensin converting enzyme inhibition capacities were lower than of those de-oiled soybean. They showed stronger DPPH radical-scavenging activity than did de-oiled soybean.     

Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain

Enterococcus faecalis UGRA10, a new AS-48-producer strain, has been isolated from a Spanish sheep’s cheese. The inhibitory substance produced by E. faecalis UGRA10 was purified and characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirming its identity with AS-48 enterocin (7.150 Da). Subsequent genetic analysis showed the existence of the as-48 gene cluster on a plasmid of approximately 70-kb. The UGRA10 strain was examined for safety properties such as enterococci virulence genes, biogenic amine production, and antibiotic resistance. As for most E. faecalis strains, PCR amplification revealed the existence of gene encoding for GelE, Asa1, Esp, EfaA, and Ace antigens and for tyrosine decarboxylase. This strain was sensitive to most of the antibiotics tested, being resistant only to aminoglycosides, lincosamide, and pristinamicins. In addition, UGRA10 developed an ability to form biofilms and to adhere to Caco 2 and HeLa 229 cells. More interestingly, this strain shows a high ability to interfere with the adhesion of Listeria monocytogenes to Caco 2 cells. Altogether, the results suggest that this broad-spectrum bacteriocin-producing strain has biotechnological potential to be developed as a protective agent in food preservation and as a probiotic.     

Heterologous expression and functional analysis of the gene cluster for the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. The gene cluster of nukacin ISK-1 consists of at least nukAMTFEG, ORF1 and ORF7. In this study, we demonstrated the heterologous production of nukacin ISK-1 in Lactococcus lactis by the artificial polycistronic expression of nukAMTFEG-ORF7 under the control of the nisin-controlled expression (NICE) system. Consequently, the recombinant L. lactis showed antimicrobial activity. Mass analysis clarified the presence of nukacin ISK-1 produced in the culture supernatant. These results suggested that the recombinant L. lactis produced nukacin ISK-1 heterologously. Inactivation of nukA, -M or -T resulted in the complete loss of the nukacin ISK-1 production phenotype. This finding suggested that nukAMT are indispensably associated with the biosynthesis of nukacin ISK-1. To our knowledge, this is the first report of the heterologous production of lantibiotic using the NICE system.     

Physico-chemical, amino acid composition, functional and antioxidant properties of roe protein concentrates obtained from Channa striatus and Lates calcarifer

Roe protein concentrates prepared from Channa striatus (CRPC) and Lates calcarifer (LRPC) were investigated for physico-chemical characteristics, amino acid composition, functional properties and antioxidant activity. Channa and Lates roes yielded 20.7% and 22.5% of protein concentrates possessing 90.2% and 82.5% protein, respectively. Major differences were not observed in each of the amino acids except leucine in CRPC and LRPC. The solubility of protein was 3.93-54.6% and 1.6-55.5% over a pH range of 2-12 in CRPC and LRPC, respectively. Water absorption, oil absorption, foam capacity, stability and emulsifying capacity were found to be higher in CRPC than in LRPC. Antioxidant activity determined by the radical scavenging activity and ferric reducing power was higher in CRPC. SDS-PAGE of both roe protein concentrates showed protein bands of 170, 95 and 55 kDa. Moisture sorption isotherms of protein concentrates indicated their hygroscopic nature.     

SB365, Pulsatilla saponin D suppresses the proliferation of human colon cancer cells and induces apoptosis by modulating the AKT/mTOR signalling pathway

Pulsatilla koreana has been used as a traditional medicine for the treatment of several diseases. The purpose of this study was to determine if SB365, Pulsatilla saponin D isolated from the root of P. koreana inhibits the progression of colon cancer. We found that SB365 strongly suppressed the growth and proliferation of colon cancer cells and induced their apoptosis. Also, SB365 showed anti-angiogenic activity by decreasing the expression of HIF-1α and VEGF. These results were confirmed by an in vivo study showing that SB365 significantly inhibited tumor growth by the induction of apoptosis and inhibition of angiogenesis with stronger anticancer activity than 5-FU. When further examined for its anticancer mechanism, SB365 effectively suppressed the AKT/mTOR pathway both in vitro and in vivo. Taken together, our study demonstrated that SB365 inhibits the AKT/mTOR pathway, leading to the suppression of tumor growth and angiogenesis together with induction of apoptosis. Therefore, SB365 is a good candidate as a natural product for use in the treatment of colon cancer.     

Varieties of antioxidant and antibacterial properties of Ecklonia stolonifera and Ecklonia kurome products harvested and processed in the Noto peninsula,Japan

Ecklonia stolonifera and Ecklonia kurome are traditional edible brown algae in the fisheries towns in Far East Asia. In the Noto Peninsula area, Ishikawa, Japan, both the Ecklonia are called ‘kajime’ and people believe that the algae improve the property of blood. To determine the varieties of antioxidant and antibacterial properties E. stolonifera and E. kurome products, assays for total phenolic content and antioxidant activities, including DPPH radical-scavenging activity, superoxide anion radical-generated by non-enzymatic system, ferrous-reducing power and WST-8, a tetrazolium salt, redox activity of four dried and two boiled E. stolonifera, and four dried and two raw E. kurome preparations were tested in this study. Furthermore, antibacterial activity of the products was tested. Though the total phenolic content, the antioxidant activity and the antibacterial activities of E. stolorifera and E. kurome products were high, these properties were varied by manufacturers or each product. Especially, two dried and two boiled products of E. stolonifera showed low activities. The results of this study indicate that the contents of functional compounds and their activities were affected or decreased greatly by the processing method.     

Apoptotic effects of cooked and in vitro digested soy on human prostate cancer cells

Previous laboratory and animal studies reported that soy isoflavones were major bioactive compounds in soy to exert chemoprotection of prostate cancer. However, these studies cannot reflect the realistic effects that soy may induce through diets, and little is known about the bioavailability of isoflavones from whole soy food and their bioactivities after cooking and digestion. In this study, cooking and in vitro digestion were used to prepare soy extracts and the effects of cooking and digestion on the isoflavone contents and bioactivities of the whole soy extracts were examined. The cooking procedure generally increased the amount of daidzin, genistin and daidzein, but decreased that of genistein. Digestion process significantly lowered contents of daidzin and genistin in 60min cooked sample, while increased the contents of daidzin and daidzein and decreased the content of genistein in the uncooked sample. Antioxidant activities of soy extracts increased after cooking and in vitro digestion, while no consistent increase of the four soy isoflavones was determined. The apoptotic effects of soy extracts on both LNCaP and C4-2B cells were generally in a dose-dependent manner. Compared to purified single isoflavones, cooked and digested soy were more effective on induction of prostate cancer cell apoptosis, which indicated synergistic interactions between various bioactive compounds in the whole soy.     

Non-toxic Salvia sclareoides Brot. extracts as a source of functional food ingredients: Phenolic profile,antioxidant activity and prion binding properties

Salvia sclareoides is an aromatic herb native to Portugal, of which phenolic content (Folin–Ciocalteau method), chemical profile (HPLC/DAD), antioxidant activity (DPPH, β-carotene/linoleic acid assays), acute toxicity (MTT method, adapted for non-adherent cells), genotoxicity (short-term chromosomal aberration assay) and prion binding properties were evaluated in the acetone, water, ethanol, methanol and n-butanol extracts. The latter presented the highest phenolic content and antioxidant activity (DPPH assay), and was the single one with the flavonoids (+)-catechin, kaempferol O-glucoside and quercetin. Vanillic acid was the major component of all extracts but gallic, gentisic, caffeic, syringic, coumaric and ferulic acids were also found in some extracts. Only the n-butanol extract had components binding to the cellular form of human prion protein detected by NMR which showed specificity for two regions of the folded domain and for the unstructured N-terminal region. Extracts were not cytotoxic nor genotoxic, reinforcing the potential of S. sclareoides for nutraceutical purposes.     

Bioactive phytochemicals, nutritional value, and functional properties of cape gooseberry (Physalis peruviana): An overview

Exotic fruits play an important role in nutrition as an excellent base for low calorie and dietetic products. Highly valued for its unique flavor, texture and color, recent research has shown cape gooseberry (Physalis peruviana) to be high in many beneficial compounds. The diversity of functional applications to which P. peruviana can be put gives this fruit great importance. The food industry has used cape gooseberry in different products including beverages, yogurts and jams. With the rapidly growing popularity of this unique fruit it is important to have a comprehensive reference for its nutritional benefits. This review provides a valuable source for current knowledge on bioactive phytochemicals and further development of P. peruviana for functional foods as well as nutraceutical and pharmaceutical industries.     

The methoxyflavones in Citrus reticulata Blanco cv. ponkan and their antiproliferative activity against cancer cells

The major polymethoxyflavones in the fruit (ponkan) peels of Citrus reticulata Blanco cv. ponkan were identified as isosinensetin, sinensetin, nobiletin and tetramethyl-o-scutellarein by a combined separation using high-speed countercurrent chromatography and preparative high performance liquid chromatography, and structure elucidation by electrospray ionisation mass spectrometry (ESI-MS) and ¹H and ¹³C nuclear magnetic resonance (NMR). The antiproliferative activity of the four compounds against four cancer cell lines (A549, HL-60, MCF-7 and HO8910) showed that isosinensetin had a lower IC₅₀ value for MCF-7 and HO8910 cancer cell lines. Determination of polymethoxyflavones in ponkan peels from different cultivation regions displayed relatively steady contents of the four compounds and a higher content of isosinensetin, which suggested that ponkan peels are excellent sources of functional polymethoxyflavones that may help prevent female cancers, such as ovarian cancer and breast cancer.     

The production of surfactin during the fermentation of cheonggukjang by potential probiotic Bacillus subtilis CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na⁺, K⁺ and Ca²⁺ ions by MALDI-TOF mass spectrometry. Upon 500 MHz ¹H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13-15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC₅₀ of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold.     

Satureja horvatii essential oil: In vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat

The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants.