Camellia sinensis tea melanin suppresses transformation of the aryl hydrocarbon receptor and prevents against dioxin-induced toxicity in mice

The suppressive effects of Camellia sinensis tea melanin (CSTM) on transformation of aryl hydrocarbon receptor (AhR) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were disclosed for the first time. CSTM strongly inhibited TCDD-induced toxicity with IC₅₀ equalling 20.4 μg mL⁻¹. Daily administration of CSTM (40 mg kg⁻¹, p.o.) prevented TCDD-induced body weight loss, ameliorated TCDD-induced mortality and prevented TCDD-induced hepatomegaly and thymic atrophy. Co-administration of CSTM significantly inhibited TCDD-induced hepatic CYP1 A1 activity. CSTM retarded transformation of AhR in vitro. In animals treated with CSTM, the antibody-secreting cells produced significantly (P < 0.05) more antibodies (32–34%) than the antigen control. Administration of TCDD caused a suppression of antibody-forming cells of 29–33% against the antigen control level. Co-administration of CSTM restored immunity to the control level. We demonstrated that CSTM directly competed with TCDD during the transformation of AhR and suppressed the downstream activation of genes associated with TCDD toxicity.     

Fractionation and identification of ACE-inhibitory peptides from α-lactalbumin and β-casein produced by thermolysin-catalysed hydrolysis

The thermolysin catalysed hydrolysates of α-lactalbumin and β-casein were fractionated by size-exclusion chromatography (SEC) and reversed-phase high performance liquid chromatography (RP-HPLC) in order to identify the peptides responsible for the high ACE-inhibitory activity of these hydrolysates. The SEC fractionation separated many co-eluting peptides into different fractions allowing individual peptides to be isolated in one or two subsequent semi-preparative RP-HPLC fractionation steps. Five potent ACE-inhibitory peptides from α-lactalbumin were isolated. They all contained the C-terminal sequence -PEW, corresponding to amino acid residues 24–26 in α-lactalbumin, and had IC₅₀ values of 1–5 μm. From one SEC fraction of the β-casein hydrolysate two potent ACE-inhibitory peptides were isolated and identified as f58-76 and f59-76 of β-casein A2. They both contained IPP as the C-terminal sequence and had IC₅₀ values of 4 and 5 μm. From another SEC fraction a new but less ACE-inhibitory peptide from β-casein was identified (f192–196; LYQQP).     

Phospholipase A2 and antioxidant enzyme activities in normal and PSE pork

In order to reveal the relationship between phospholipase A₂ and antioxidant enzymes and drip loss in pork, the study was designed to examine the effects of phospholipase A₂ and antioxidant enzymes on the water-holding capacity of pork during postmortem chilling. Six PSE and RFN samples (longissimus muscle) were used to determine the activities of phospholipase A₂ (tPLA_2, cPLA₂ + sPLA₂ and iPLA₂), superoxide dismutase (SOD) and GSH-Px, and acid phospholipase. The results showed that pH_1 h and pH_24 h from PSE pork were lower (p < 0.01) than for normal pork (RFN), but the L* value at 1 h and 24 h postmortem, TBARS content, drip loss at 48 h and 96 h, cooking loss, tPLA₂ activity and iPLA₂ were higher (p < 0.01) than of normal pork. Correlation analysis indicated that drip loss at 48 h was negatively related to pH_1 h (p < 0.01) and pH_24 h (p < 0.01) but positively to T_1 h (p < 0.01) and the activities of total phospholipase A₂ (p < 0.05) and calcium-independent phospholipases A₂ (p < 0.01). The tPLA₂ and GSH-Px play important roles in drip loss.     

Indolylfuran,a potent aryl hydrocarbon receptor agonist from sauerkraut,interacts with the oestrogen pathway

Numerous ligands of the aryl hydrocarbon receptor (AhR) have been found in plants, especially edible plants, such as cruciferous vegetables, which exert beneficent health effects. A potent activator of the AhR was found in sauerkraut juice. The isolated active ingredient was identified as the novel AhR ligand, (1-(2-furanyl)2-(3-indolyl)ethanone, common name indolylfuran. The isolated and the synthesised compound exerted similar potencies; their EC₅₀-values in an AhR transactivation assay were 160 and 123 nM, respectively. Our in vivo studies confirm and enlighten basic interactions between the AhR and oestrogen receptors (ERs). Further anti-oestrogenic effects of sauerkraut extract were shown. Indolylfuran regulates ER α and β expression, most likely via the AhR pathway, since indolylfuran had no effect on uterus weight and did not agonise ERα. Sauerkraut and indolylfuran may have potential for the prevention or treatment of diseases through modulation of AhR regulation and, indirectly, the ER pathway.     

Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells

RL95-2 endometrial cancer cells were used to study cytochrome P450-mediated chemopreventative mechanisms of four flavonoids found in foods. To investigate enzymatic CYP1 inhibition, intact cells were induced with benzo(a)pyrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Quercetin, kaempferol and myricetin inhibited CYP1 activity dose-dependently with IC₅₀s ranging from 2.2 to 4 μM; while amentoflavone was inactive. Further experiments were designed to determine if flavonoids also interacted with the AhR or caused a decrease in CYP1 protein or mRNA expression. CYP1A1 protein expression was inhibited in cells co-treated with TCDD and quercetin, kaempferol or myricetin compared with TCDD alone, but amentoflavone was ineffective. Relatively higher (∼7-fold) basal levels of CYP1B1 protein were not significantly affected by flavonoid treatments. In general, at the message level significant inhibition of induced CYP1A1 or CYP1B1 was not detected following flavonoid cotreatment. Despite the common inhibitory effects of quercetin, kaempferol, and myricetin on induced CYP1A1-dependent activity and protein expression, the mechanisms of CYP1 inhibition in this cell line are complex and dependent on the CYP gene, AhR inducer and the flavonoid.     

Radiation-inducible TNF-α gene expression under stress-inducible promoter gadd 153 for cancer therapy

We demonstrated the effectiveness of radiation-inducible expression of the TNF-α gene for cancer therapy in vitro. The TNF-α gene under the control of the stress-inducible promoter, gadd 153, was introduced into the human glioma cell line, U251-SP. Without cobalt-60 gamma irradiation, no cytotoxicity against the transfected cells was observed. When the transfected cells were irradiated with 10 or 20 gray (Gy), the gadd 153 promoter was highly induced and the expression level of TNF-α increased. Five days after the irradiation, the TNF-α productions of each cell irradiated with 10 and 20 Gy were 30 and 100 times higher than the basal level, respectively. The cytotoxicities against the transfected cells 5 d after irradiation with 10 and 20 Gy were 79% or 91%, respectively, which are much higher than those against the nontransfected cells that were irradiated at the same dose (43% and 78%, respectively). These results demonstrate that the gadd 153-TNF-α system may be an effective tool for radiosurgery of malignant brain tumors.     

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B₁ (AFB₁) by white rot fungi through laccase production was investigated in different liquid media. A significant (P < 0.0001) correlation was observed between laccase activity and AFB₁ degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r = 0.93) and mineral salts — malt extract (MSB–MEB) (r = 0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB–MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496 U/L), as well as 40.45% AFB₁ degradation as monitored using high performance liquid chromatography. P. ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39 U/L and 35.90% degradation of AFB₁. Aflatoxin B₁ was significantly (P < 0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1 U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118 U/L, 55%). Aflatoxin B₁ degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P < 0.001) loss of mutagenicity of AFB₁, as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB₁ by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.     

Theasaponin E1 destroys the salt tolerance of yeasts

Cells of Zygosaccharomyces rouxii in a medium containing a high concentration of NaCl were killed during incubation for 2–4 h with a low concentration of a mixture of saponins from tea seeds (TSS). The higher the concentration of NaCl in the medium, the higher the inhibitory effect of TSS on the growth of the yeast. The above inhibitory effect of TSS on the growth of the yeast was not observed when cells were incubated in hypertonic media composed of nonionic substances such as sugars. The ATPase activity of plasma membrane preparations from the yeast cells was slightly affected by the addition of TSS. It is shown that TSS facilitates leakage of glycerol from the yeast cells under NaCl-hypertonic conditions. The major inhibitor in the mixture of saponins was isolated and identified as theasaponin E₁. Its isomer, theasaponin E₂, did not have any effect on the salt tolerance of Z. rouxii or Saccharomyces cerevisiae.     

Lunasin induces apoptosis and modifies the expression of genes associated with extracellular matrix and cell adhesion in human metastatic colon cancer cells

Scope: Lunasin is an arginine‐glycine‐aspartic acid (RGD) cancer preventive peptide. The objective was to evaluate the potential of lunasin to induce apoptosis in human colon cancer cells and their oxaliplatin‐resistant (OxR) variants, and its effect on the expression of human extracellular matrix and adhesion genes. Methods and results: Various human colon cancer cell lines which underwent metastasis were evaluated in vitro using cell flow cytometry and fluorescence microscopy. Lunasin cytotoxicity to different colon cancer cells correlated with the expression of α₅b₁ integrin, being most potent to KM12L4 cells (IC₅₀ = 13 μM). Lunasin arrested cell cycle at G2/M phase with concomitant increase in the expression of cyclin‐dependent kinase inhibitors p21 and p27. Lunasin (5–25 μM) activated the apoptotic mitochondrial pathway as evidenced by changes in the expressions of Bcl‐2, Bax, nuclear clusterin, cytochrome c and caspase‐3 in KM12L4 and KM12L4‐OxR. Lunasin increased the activity of initiator caspase‐9 leading to the activation of caspase‐3 and also modified the expression of human extracellular matrix and adhesion genes, downregulating integrin α₅, SELE, MMP10, integrin β₂ and COL6A1 by 5.01‐, 6.53‐, 7.71‐, 8.19‐ and 10.10‐fold, respectively, while upregulating COL12A1 by 11.61‐fold. Conclusion: Lunasin can be used in cases where resistance to chemotherapy developed.     

Reduction of soybean oligosaccharides and properties of alpha-D-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteroides [corrected] JK55

This study was undertaken to investigate the potential for reducing non-digestive oligosaccharides (NDO) in soy foods, as well as the influence of exogenous conditions on intracellular α-galactosidase (α-Gal) producing lactic acid bacteria. Two strains, Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55, showed the highest levels of raffinose degrading activity at over 40 U mL⁻¹, and presented maximum activities during the stationary phase in a medium where raffinose was the only carbon source. Raffinose was the most effective inducer, followed by melibiose, and galactose; the enzymes were partially inhibited by fructose and sucrose. On the other hand, limited activity was observed in glucose. The strains displayed optimum activity levels at neutral pH and a 35–37 °C temperature range. The α-Gal activities of L. curvatus R08 and Leu. mesenteriodes JK55 were maintained at pH 6.5–10.0. The activity of the α-Gal enzyme was stable in a relatively broad range of temperatures from 0 to 40 °C for 3 h. In soymilk, Leu. mesenteriodes JK55 and L. curvatus R08 completely hydrolyzed the NDO after 18–24 h of fermentation. The abilities of L. curvatus R08 and Leu. mesenteriodes JK55 to degrade raffinose sugars and, particularly, to produce organic acids from sugar, could contribute to reductions in the anti-nutritional properties of soy, and to the accumulation of compounds with beneficial properties during food processing. Furthermore, this study provides the optimum conditions to induce α-Gal from these strains.     

Heat-adaptation induced thermotolerance in Staphylococcus aureus: Influence of the alternative factor σ

The role of σ^B in the Staphylococcus aureus heat-shock induced thermotolerance was investigated. Survival curves at 58 °C of S. aureus strain Newman and its isogenic ΔsigB mutant were obtained for native and heat-shocked cells (45 °C for 5–120 min) in exponential and stationary phase of growth. The magnitude of the acquisition of thermotolerance at 58 °C depended on the growth phase and on the duration of the heat shock. Stationary growth phase cells were always more heat tolerant than exponentially growing cells and thermotolerance increased with heat-shock duration up to 120 min. S. aureus cells were able to increase their heat tolerance in the absence of the σ^B factor. In stationary phase, whereas in the parental strain the thermotolerance was increased by a factor of 12 after a heat shock of 120 min at 45 °C (δ values at 58 °C for native and heat-shocked cells were 0.63 and 7.22 min, respectively), in the mutant strain it increased 43 fold (δ values 0.09 and 3.87 min). The addition of chloramphenicol to the adaptation medium resulted in a lower increase in heat tolerance but did not prevent it completely, suggesting that S. aureus can partially increase its thermotolerance without “de novo” protein synthesis. Both the number of non-damaged cells and the proportion of cells able to repair sublethal damage were higher for heat-shocked cells.     

Bioactivity of Ocimum gratissimum L. oil and two of its constituents against five insect pests attacking stored food products

The fumigant and repellent effects of Ocimum gratissimum L. oil and its constituents, β-(Z)-ocimene and eugenol, were evaluated against adults of Sitophilus oryzae (L.), Tribolium castaneum (Herbst), Oryzaephilus surinamensis (L.), Rhyzopertha dominica (F.) and Callosobruchus chinensis (L.). The fumigant toxicities of the oil and two of its constituents were assessed at four rates (0, 1, 5 and10 μL/L air) in space fumigation, whereas repellence of the oil and eugenol in acetone was evaluated in choice bioassays at five rates (0, 1.0, 2.0, 3.0 and 4.0 μL oil/2 g grain). Results showed that fumigant toxicity and repellence of the oil and its constituents were significantly (P<0.0001) influenced by concentration and time after treatment. At 1 μL/L air, the oil caused 98%, 99% and 100% mortality of R. dominica, O. surinamensis and C. chinensis, respectively, 24 h after treatment, whereas eugenol achieved 79%, 61% and 100% kill of the same insects. β-(Z)-ocimene produced a weaker toxicity with 8%, 11% and 59% mortality of R. dominica, O. surinamensis and C. chinensis, respectively. Except for T. castaneum which was more tolerant, LC₅₀ values for tested insects ranged from 0.20 to 14, 0.01 to 17 and 0.80 to 23 μL/L air 24 h after treatment for O. gratissimum oil, eugenol and β-(Z)-ocimene, respectively. All test insects had percentage repellence (PR) values which ranged from 37.5% to 100% and 45% to 100% for the oil and eugenol, respectively. However, C. chinensis showed a dose-dependent decrease in PR values in the eugenol bioassay with a corresponding dose-dependent mortality on treated grains. Ocimum gratissimum oil and its constituents are potential alternatives to synthetic fumigants in the treatment of durable agricultural products. Successful adoption of plant oils in the protection of food commodities promises an eco-friendly option compatible with international biosafety regulations.     

Chemical Constituents of Gold‐red Apple and Their α‐Glucosidase Inhibitory Activities

Ten compounds were isolated and purified from the peels of gold‐red apple (Malus domestica) for the 1st time. The identified compounds are 3β, 20β‐dihydroxyursan‐28‐oic acid (1), 2α‐hydroxyoleanolic acid (2), euscaphic acid (3), 3‐O‐p‐coumaroyl tormentic acid (4), ursolic acid (5), 2α‐hydroxyursolic acid (6), oleanolic acid (7), betulinic acid (8), linolic acid (9), and α‐linolenic acid (10). Their structures were determined by interpreting their nuclear magnetic resonance and mass spectrometry (MS) spectra, and by comparison with literature data. Compound 1 is new, and compound 2 is herein reported for the 1st time for the genus Malus. α‐Glucosidase inhibition assay revealed 6 of the triterpenoid isolates as remarkable α‐glucosidase inhibitors, with betulinic acid showing the strongest inhibition (IC₅₀ = 15.19 μM). Ultra‐performance liquid chromatography‐electrospray ionization MS analysis of the fruit peels, pomace, flesh, and juice revealed that the peels and pomace contained high levels of triterpenes, suggesting that wastes from the fruit juice industry could serve as rich sources of bioactive triterpenes.     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Identification and characterization of Dekkera bruxellensis, Candida pararugosa, and Pichia guilliermondii isolated from commercial red wines

Yeast isolates from commercial red wines were characterized with regards to tolerances to molecular SO₂, ethanol, and temperature as well as synthesis of 4-ethyl-phenol/4-ethyl-guaiacol in grape juice or wine. Based on rDNA sequencing, nine of the 11 isolates belonged to Dekkera bruxellensis (B1a, B1b, B2a, E1, F1a, F3, I1a, N2, and P2) while the other two were Candida pararugosa (Q2) and Pichia guilliermondii (Q3). Strains B1b, Q2, and Q3 were much more resistant to molecular SO₂ in comparison to the other strains of Dekkera. These strains were inoculated (10³–10⁴ cfu/ml) along with lower populations of Saccharomyces (<500 cfu/ml) into red grape juice and red wine incubated at two temperatures, 15 °C and 21 °C. Although Saccharomyces quickly dominated fermentations in grape juice, B1b and Q2 grew and eventually reached populations >10⁵ cfu/ml. In wine, Q3 never entered logarithmic growth and quickly died in contrast to Q2 which survived >40 days after inoculation. B1b grew well in wine incubated at 21 °C while slower growth was observed at 15 °C. Neither Q2 nor Q3 produced 4-ethyl-phenol or 4-ethyl-guaiacol, unlike B1b. However, lower concentrations of volatile phenols were present in wine incubated at 15 °C compared to 21 °C.     

Partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra)

The partial hydrolysis of soybean oil, as catalysed by phospholipase A₁ (Lecitase Ultra) in a solvent-free system, was investigated in this study. The optimal pH and temperature for the partial hydrolysis of soybean oil by phospholipase A₁ (Lecitase Ultra) were 6.8 and 40 °C, respectively. Phospholipase A₁ (Lecitase Ultra) displayed good stability over a range of pH values from 4.7 to 7.4, and at temperatures below 60 °C. Phospholipase A₁ (Lecitase Ultra) is postulated to possess sn-1,3-position regiospecificity towards triacylglycerols (TAGs), based on the identification of the fatty acids released from TAGs following partial hydrolysis by swine pancreatic lipase (SPL) and phospholipase A₁ (Lecitase Ultra). Alternative TAG hydrolysis routes for phospholipase A₁ (Lecitase Ultra) are postulated, and several reaction equilibrium and rate constants were determined.