Synergistic Antitumor Effect of Oligogalacturonides and Cisplatin on Human Lung Cancer A549 Cells.

Cisplatin (DPP), a clinically potent antineoplastic agent, is limited by its severe adverse effects. The aim of this study was to investigate the effect of oligogalacturonides (OGA) and DDP on human lung cancer A549 cells. The combined use of OGA and DDP had a synergistic effect on the growth inhibition of A549 cells, changed the cell cycle distribution, and enhanced apoptotic response, especially in sequential combination treatment group of DDP 12 h + OGA 12 h. Western blot analyses showed that the combination treatment of OGA and DDP upregulated Bax, p53, and Caspase-3 and downregulated Bcl-2 proteins. More importantly, DDP-induced toxicity was attenuated by OGA and DDP combination treatment in normal HEK293 cells. Our data suggests that the combined use of OGA from natural sources and DDP could be an important new adjuvant therapy for lung cancer as well as offer important insights for reducing kidney toxicity of DDP and delaying the development of DDP resistance.

Protective Effect of Vanillic Acid against Hyperinsulinemia, Hyperglycemia and Hyperlipidemia via Alleviating Hepatic Insulin Resistance and Inflammation in High-Fat Diet (HFD)-Fed Rats.

Excess free fatty acid accumulation from abnormal lipid metabolism results in the insulin resistance in peripheral cells, subsequently causing hyperinsulinemia, hyperglycemia and/or hyperlipidemia in diabetes mellitus (DM) patients. Herein, we investigated the effect of phenolic acids on glucose uptake in an insulin-resistant cell-culture model and on hepatic insulin resistance and inflammation in rats fed a high-fat diet (HFD). The results show that vanillic acid (VA) demonstrated the highest glucose uptake ability among all tested phenolic acids in insulin-resistant FL83B mouse hepatocytes. Furthermore, rats fed HFD for 16 weeks were orally administered with VA daily (30 mg/kg body weight) at weeks 13-16. The results show that levels of serum insulin, glucose, triglyceride, and free fatty acid were significantly decreased in VA-treated HFD rats (p < 0.05), indicating the protective effects of VA against hyperinsulinemia, hyperglycemia and hyperlipidemia in HFD rats. Moreover, VA significantly reduced values of area under the curve for glucose (AUCglucose) in oral glucose tolerance test and homeostasis model assessment-insulin resistance (HOMA-IR) index, suggesting the improving effect on glucose tolerance and insulin resistance in HFD rats. The Western blot analysis revealed that VA significantly up-regulated expression of hepatic insulin-signaling and lipid metabolism-related protein, including insulin receptor, phosphatidylinositol-3 kinase, glucose transporter 2, and phosphorylated acetyl CoA carboxylase in HFD rats. VA also significantly down-regulated hepatic inflammation-related proteins, including cyclooxygenase-2 and monocyte chemoattractant protein-1 expressions in HFD rats. These results indicate that VA might ameliorate insulin resistance via improving hepatic insulin signaling and alleviating inflammation pathways in HFD rats. These findings also suggest the potential of VA in preventing the progression of DM.

Assessment of oligogalacturonide from citrus pectin as a potential antibacterial agent against foodborne pathogens.

Foodborne diseases are an important public health problem in the world. The bacterial resistance against presently used antibiotics is becoming a public health issue; hence, the discovery of new antimicrobial agents from natural sources attracts a lot of attention. Antibacterial activities of oligogalacturonide from commercial microbial pectic enzyme (CPE) treated citrus pectin, which exhibits antioxidant and antitumor activities, against 4 foodborne pathogens including Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Pseudomonas aeruginosa was assessed. Pectin hydrolysates from CPE hydrolysis exhibited antibacterial activities. However, no antibacterial activity of pectin was observed. Citrus oligogalacturonide from 24-h hydrolysis exhibited bactericidal effect against all selected foodborne pathogens and displayed minimal inhibitory concentration at 37.5 µg/mL for P. aeruginosa, L. monocytogenes, and S. Typhimurium, and at 150.0 µg/mL for S. aureus.

Hepatoprotective effect of silymarin on di(2-ethylhexyl)phthalate (DEHP) induced injury in liver FL83B cells.

Di(2-ethylhexyl)phthalate (DEHP), is a toxic environmental pollutant in our life which can contaminate air, water, and soil. The hepatoprotective effect of silymarin on DEHP-induced injury in FL83B mouse liver cells was investigated by analyzing the cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), cell cycle arrest, and cell morphology. The results revealed that cell viability decreased while released LDH and ALT increased with the increase of DEHP concentrations. Moreover, cell population of sub-G1 and S phase increased as the concentrations of DEHP increased. Silymarin at 25 µM achieved the highest hepatoprotective effect and exhibited 79% cell viability while only 46% cell viability was found in DEHP injured control. It was also found to reduce LDH release and cell populations of sub-G1 and S phase. Therefore, silymarin could ameliorate DEHP-induced injury and have potential to be further developed as a natural ingredient of health food against phthalate plasticizers induced liver injury.

Pectin methyl esterase treatment on high-methoxy pectin for making fruit jam with reduced sugar content.

BACKGROUND: Pectin methyl esterase (PME) has been postulated to catalyse the transacylation reaction between pectin molecules. The present study aimed to prove the occurrence of this reaction. The feasibility of applying PME-catalysed transacylation between high-methoxy pectin molecules in making fruit jam with reduced sugar content was also investigated. RESULTS: PME treatment increased the turbidity and particle size in pectin solution and the molecular weight of pectin, while it decreased the number of methoxy ester linkages and the intensity of the CH3 absorption peak in the Fourier transform infrared spectrum without changes in the number of total ester linkages in pectin molecules. These findings support the occurrence of PME-catalysed transacylation between pectin molecules. Higher values of hardness, gumminess and chewiness were found in a jam containing PME-treated citrus pectin (10 g L⁻¹) and sugar (350 g L⁻¹) as compared with either a jam containing untreated citrus pectin (10 g L⁻¹) and sugar (350 g L⁻¹) or strawberry jam containing pectin (10 g L⁻¹) from the fruit and sugar (650 g L⁻¹). CONCLUSION: The demand for sugar in jam making can be greatly reduced by the use of PME-treated high-methoxy pectin.

Antioxidant activity and emulsion-stabilizing effect of pectic enzyme treated pectin in soy protein isolate-stabilized oil/water emulsion.

The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-ß-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.

In vitro antitumor and immunomodulatory effects of the protein PCP-3A from mushroom Pleurotus citrinopileatus.

A nonlectin glycoprotein (PCP-3A) newly isolated from the fruit body of edible golden oyster mushroom Pleurotus citrinopileatus has been shown to be growth inhibitory against human myeloid leukemic U937 cells in a previous report. There is a well-recognized relation between antitumor activity and immunomodulation. The immunomodulatory activity of PCP-3A was therefore assessed in the present study. Human mononuclear cells (MNC) and the CD4(+) T lymphocytes isolated from them were stimulated separately with PCP-3A for various durations and then filtered to obtain the conditioned media (CM). The conditioned medium from MNC (MNC-CM) was proved effective in inhibiting the growth of U937 cells. Increased secretion of cytokines TNF-α, IL-2, and IFN-γ from the stimulated MNC and CD4(+) T cells was found in CM. The antibody neutralization test of MNC-CM revealed that the growth inhibition on leukemic U937 cells was related to the elevation in cytokine concentration. We propose that PCP-3A stimulated human MNC to secrete cytokines TNF-α, IL-2, and IFN-γ, which subsequently inhibit the growth of U937 cells, and that PCP-3A may be a possible material for developing into an antileukemia ingredient in health food.

A glycoprotein extracted from golden oyster mushroom Pleurotus citrinopileatus exhibiting growth inhibitory effect against U937 leukemia cells.

Mushrooms have become popular sources of natural antitumor, antiviral, antibacterial, antioxidative, and immunomodulatory agents. Golden oyster mushroom, Pleurotus citrinopileatus , is a common mushroom in oriental countries for human consumption. We isolated a functional protein (PCP-3A) from the fresh fruiting body of this mushroom. The isolation procedure included ammonium sulfate fractionation, DEAE-Sepharose CL-6B ion exchange chromatography, and Sephacryl S-300 gel filtration. Electrophoresis demonstrated that PCP-3A is a glycoprotein composed of 10 subunits, each approximately 45.0 kDa in size. In vitro cell study showed that PCP-3A at a concentration about 12.5 microg/mL inhibits the proliferation of human tumor cell line U937, in a time- dependent manner (24, 48, and 72 h). It failed to agglutinate rabbit and human erythrocytes, excluding its possibility from being a lectin. Flow cytometry revealed that it is capable of inhibiting the growth of U937 cells by way of S phase arrest and apoptotic induction. We suggest that PCP-3A is worth further investigating for antitumor use.

Antimutagenic and antiproliferative effects of roasted and defatted peanut dregs on human leukemic U937 and HL-60 cells.

The antimutagenic effects on Salmonella typhimurium TA98 and TA100 strains and antiproliferative effects on leukemia cell lines (U937 and HL-60) of peanut protein isolate (PPI), peanut protein isolate enzyme hydrolysate (PPIEH), roasted and defatted peanut dregs (RDPD), and roasted and defatted peanut dregs enzyme hydrolysate (RDPDEH) were investigated. The antimutagenic effects on B(a)P and 4-NQO toward the TA98 and TA100 strains were found to follow a diminishing order: RDPD > RDPDEH >> PPI = PPIEH with dose-dependency. Antiproliferative effects on leukemia cells U937 and HL-60 were also detected. RDPD was found to be the most effective of all the peanut preparations. At 100 microg/mL concentration, RDPD inhibited the proliferation of U937 and HL-60 cells by 56% and 52%, respectively. We propose to consider RDPD and RDPDEH in the development of natural chemotherapeutic or chemopreventive dietary supplements against leukemia and to upgrade the utilization of these by-products in peanut oil production.

Separation and utilization of pectin lyase from commercial pectic enzyme via highly methoxylated cross-linked alcohol-insoluble solid chromatography for wine methanol reduction.

The isolation and utilization of pectin lyase (PL) from commercial pectic enzyme for methanol reduction in wine production was investigated. PL can be separated from pectinesterase (PE) and polygalacturonase (PG) on HM-CL-AIS affinity chromatography at pH 4; however, it is difficult to further distinguish PE from PG. Some desirable physicochemical properties such as transmittance, lightness, redness, and lower total pectin content are found in the external enzyme adding groups (PL, PE and PG, and pectic enzyme groups) in comparison to the control group. Methanol contents in pectic enzyme and the PE and PG groups increase from 628 +/- 13 (control group) to 3103 +/- 16 and 1736 +/- 67 mg/L ethanol in the final products, respectively. Nevertheless, the adding of PL does not cause any increase in methanol content. The results present in this study suggest that the HM-CL-AIS column is a simple, inexpensive, convenient, and effective method for PL purification. Moreover, the partial purified PL is a potential replacement of commercial pectic enzyme for pectin depolymerizing, methanol content reducing, and wine quality improving in wine production.

Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

Structural and functional characterizations of mung bean mitochondrial nucleoids.

Mitochondrial nucleoids isolated from mung bean seedlings exhibited a chromatin-like structure associated with a membrane component. A similar structure, which underwent discrete changes during cotyledon development, was identified in situ. Isolated nucleoids consisted of essentially the same phospholipids, including cardiolipin, as whole mitochondria and proteins of inner- and outer-mitochondrial-membrane origin. Actin was consistently found with mitochondrial nucleoids prepared with different detergent concentrations. Formaldehyde cross-linking of cytochalasin B- and proteinase K-treated mitochondria further revealed that actin was associated with DNA in nucleoids. Mitochondrial nucleoids were self-sufficient in directing DNA synthesis in vitro in a pattern mimicking mtDNA synthesis in isolated mitochondria. In pulse-field gel electrophoresis, newly synthesized mtDNA separated into two major components, well-bound and fast-moving forms. Nucleoids DNA synthesis was resistant to aphidicolin but sensitive to N-ethylmaleimide, which indicates that a gamma-type DNA polymerase was responsible for this activity. Mitochondrial nucleoids were capable of self-directed RNA transcription in a non-random fashion in vitro. Consistent with and complementary to results from fungi and human cells done mostly in situ, our present work helps to establish the important paradigm that mitochondrial nucleoids in eukaryotes are more than mere mtDNA compaction and segregation entities but are centers of mtDNA maintenance and expression.

Pectinesterase inhibitor from jelly fig (Ficus awkeotsang Makino) achene induces apoptosis of human leukemic U937 cells.

The antitumor activity of pectinesterase inhibitor (PEI), a group of cationic polypeptides, from jelly fig (Ficus awkeotsang Makino) achene was first examined as a treatment for leukemia in this study. PEI displayed strong growth inhibition against human leukemic U937 cells via induction of apoptosis in a dose- and time-dependent manner. At a level of 50 microg/mL, PEI inhibited 90% of cell growth, and the concentration of PEI required to induce 50% of cell viability (LC50) was about 180 microg/mL. Meanwhile, cell cycle arrest at G2/M phase was observed when cells were incubated with 100 microg PEI/mL for 24 h. PEI displayed a dose-dependent influence on mitochondria transmembrane potential (MTP, delta psi m) of cells when detected by a flow cytometry. MTP of more than 50% cells was reduced when cells were incubated with PEI at levels higher than 50 microg PEI/mL for 24 h. In addition, PEI upregulated caspase-3 activity. Taken together, PEI potently inhibited the proliferation of human leukemic U937 cells via cell cycle arrest and apoptosis in association with MTP reduction and caspase-3 activation, respectively, and showed therapy potential for U937 cells.

Characterization of senescence-associated proteases in postharvest broccoli florets.

We characterized the senescence-associated proteases of postharvest broccoli (Brassica oleracea L. var Green King) florets, using class-specific protease inhibitors and gelatin-polyacrylamide gel electrophoresis. Different classes of senescence-associated proteases in broccoli florets were partially characterized for the first time. Protease activity of broccoli florets was depressed by all the inhibitors and showed different inhibition curves during postharvest. The hydrolytic activity of metalloprotease (EC 3.4.24. - ) and serine protease (EC 3.4.21. - ) reached a maximum, 1 day after harvest (DAH), then decreased, while the hydrolytic activity of cysteine protease (EC 3.4.22. - ) and aspartic protease (EC 3.4.23. - ) increased throughout the postharvest senescence based on the calculated inhibition percentage of protease activity. The senescence-associated proteases were separated into seven endoprotease (EP) groups by gelatin-polyacryamide gel electrophoresis and classified into EP1 (metalloprotease), EP2 (metalloprotease and cysteine protease), EP3 (serine protease and aspartic protease), EP4, EP5, EP7 (cysteine protease), and EP6 (serine protease) based on the sensitivity of class-specific protease inhibitors. The proteases EP2, EP3, and EP4 were present throughout the postharvest stages. EP3 was the major EP at all times during senescence; EP4 intensity of activity increased after 2 DAH; EP6 and EP7 clearly increased after 4 DAH. Our results suggest that serine protease activity contributes to early stage (0-1 DAH) and late stage (4-5 DAH) of senescence; metalloprotease activity was involved in the early and intermediate stages (0-3 DAH) of senescence; and cysteine protease and aspartic protease activities participated in the whole process of broccoli senescence.

Novel broccoli 1-aminocyclopropane-1-carboxylate oxidase gene (Bo-ACO3) associated with the late stage of postharvest floret senescence.

A novel 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene (Bo-ACO3) was first isolated from senescing broccoli florets by subtractive hybridization. The cDNA clone comprised a 963 bp open reading frame encoding a protein of 321 amino acids. The predicted molecular mass and pI were 36 kDa and 5.42, respectively. Bo-ACO3 shares 68% identity in the coding region with Bo-ACO1 (ACC Ox1) and Bo-ACO2 (ACC Ox2) and is quite divergent from the 3' untranslated regions. Bo-ACO3 transcript was accumulated to high levels only at the late stage of senescence after harvest. Southern blot hybridization using full-length cDNA as a probe suggested that the Bo-ACO3 gene is a single-copy gene in the broccoli genome. The deduced 321 amino acid sequence of Bo-ACO3 shares 70% identity with either Bo-ACO1 or Bo-ACO2. The BO-ACO3 gene was expressed in Escherichia coli as a 38 kDa active ACO enzyme. It was concluded that Bo-ACO3 is a senescence-associated gene involved in the late-phase senescence of postharvest broccoli.