Preparation of carotenoid extracts and nanoemulsions from Lycium barbarum L. and their effects on growth of HT-29 colon cancer cells.

Lycium barbarum L., a traditional Chinese herb widely used in Asian countries, has been demonstrated to be protective against chronic diseases such as age-related macular degeneration. The objectives of this study were to determine the carotenoid content in L. barbarum by high-performance liquid chromatography-mass spectrometry, followed by preparation of a carotenoid nanoemulsion to evaluate the mechanism of inhibition on HT-29 colon cancer cells. The highest extraction yield of carotenoids was attained by employing a solvent system of hexane-ethanol-acetone (1:1:1, v/v/v). Nine carotenoids, including neoxanthin (4.47 µg g-1), all-trans-zeaxanthin and its cis-isomers (1666.3 µg g-1), all-trans-ß-cryptoxanthin (51.69 µg g-1), all-trans-ß-carotene and its cis-isomers (20.11 µg g-1), were separated within 45 min and quantified using a YMC C30 column and a gradient mobile phase of methanol-water (9:1, v/v) (A) and methylene chloride (B). A highly stable carotenoid nanoemulsion composed of CapryolTM 90, Transcutol®HP, Tween 80 and deionized water was prepared with a mean particle size of 15.1 nm. Characterization of zeaxanthin standard, blank nanoemulsion, carotenoid extract and carotenoid nanoemulsion by differential scanning calorimetry curves and Fourier transform infrared spectra revealed a good dispersion of zeaxanthin-dominated carotenoid extract with no significant chemical change after incorporation into nanoemulsion. The in vitro release kinetic study showed a higher release profile at pH 5.2 than at physiological pH 7.4, suggesting a rapid release of carotenoids in the acidic environment (pH 4.5-6.5) characteristic of tumors. Both the carotenoid nanoemulsion and the extract were effective at inhibiting growth of HT-29 colon cancer cells, with an IC50 of 4.5 and 4.9 µg ml-1, respectively. Also, both treatments could up-regulate p53 and p21 expression and down-regulate CDK2, CDK1, cyclin A and cyclin B expression and arrest the cell cycle at G2/M. The study may form a basis for further exploration of L. barbarum nanoemulsion in cancer treatment.

The synthesis and characterization of poly(γ-glutamic acid)-coated magnetite nanoparticles and their effects on antibacterial activity and cytotoxicity.

Magnetite nanoparticles (MNPs) modified with sodium and calcium salts of poly(γ-glutamic acid) (NaPGA and CaPGA) were synthesized by the coprecipitation method, followed by characterization and evaluation of their antibacterial and cytotoxic effects. Superparamagnetic MNPs are particularly attractive for magnetic driving as well as bacterial biofilm and cell targeting in in vivo applications. Characterization of synthesized MNPs by the Fourier transform infrared spectra and magnetization curves confirmed the PGA coating on MNPs. The mean diameter of NaPGA- and CaPGA-coated MNPs as determined by transmission electron microscopy was 11.8 and 14 nm, respectively, while the x-ray diffraction pattern revealed the as-synthesized MNPs to be pure magnetite. Based on agar dilution assay, both NaPGA- and CaPGA-coated MNPs showed a lower minimum inhibitory concentration in Salmonella enteritidis SE 01 than the commercial antibiotics linezolid and cefaclor, but the former was effective against Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 10832, whereas the latter was effective against Escherichia coli O157:H7 TWC 01. An in vitro cytotoxicity study in human skin fibroblast cells as measured by MTT assay implied the as-synthesized MNPs to be nontoxic. This outcome demonstrated that both γ-PGA-modified MNPs are cytocompatible and possess antibacterial activity in vitro, and thereby should be useful in in vivo studies for biomedical applications.

Anti-inflammatory effects of isoflavone powder produced from soybean cake.

Soybean cake, a byproduct obtained during the processing of soybean oil, has been shown to be a rich source of isoflavones. The objectives of this study were to use soybean cake as raw material for processing into powder and to evaluate the anti-inflammatory activity. Eleven treatments, including powders of malonylglucoside, glucoside, acetylglucoside, aglycone, ISO-1, and ISO-2, as well as genistein standard, gamma-PGA, control, normal, and PDTC, were used for evaluation. A total of 77 mice were each provided daily with tube feeding for 4 weeks at a dose of 0.3 mL of aqueous solution from each treatment, and inflammation was induced with intraperitoneal injection of 1 mg/kg of body weight lipopolysaccharide (LPS). Results showed that all of the isoflavone powders and genistein standard were effective in inhibiting LPS-induced inflammation, lowering leukocyte number in mice blood and reducing production of IL-1beta, IL-6, NO, and PGE2 in both peritoneal exudate cell supernatant and peritoneal exudate fluid. All of the isoflavone treatments failed to retard T cell proliferation; however, both ISO-1 and ISO-2 could inhibit B cell proliferation. The difference in anti-inflammatory activity was minor between any of the isoflavone treatments.

Interaction of PRK1 receptor-like kinase with a putative elF2B beta-subunit in tobacco.

PRK1, a receptor-like kinase that is expressed in pollen, pollen tubes, and ovaries, has been shown to play important roles in pollen development and embryo sac development in Petunia inflata. We have used the kinase domain of PRK1 as a bait in the yeast two-hybrid system to identify PRK1-interacting proteins. The screening resulted in isolation of a cDNA encoding a protein highly homologous to the human and yeast beta-subunit of translation initiation factor 2B (eIF2B-beta), which was designated NeIF2Bbeta. eIF2B is a guanine nucleotide exchange protein that functions in the regulation of translation in eukaryotic cells. Deletion mutants of NeIF2Bbeta were analyzed for their interaction with PRK1, and the results suggested that the N-terminal half of NeIF2Bbeta, especially the region between residue 103 and 235, is important for the interaction. This protein association was confirmed by in vitro binding assay of the recombinant NeIF2Bbeta and PRK1 proteins. Despite high sequence homology between NeIF2Bbeta and its yeast counterpart, the NeIF2Bbeta cDNA could not rescue the phenotype of the yeast mutant strain lacking the GCD7 gene encoding eIF2B-beta, when transferred into the mutant strain.

Effects of heating time and antioxidants on the formation of heterocyclic amines in marinated foods.

The effect of heating time and antioxidants on the heterocyclic amine (HAs) formation in marinated foods were studied. Food samples were cooked at 98 +/- 2 degrees C for 1, 2, 4, 8, 16 and 32 h in a closed pan in the presence of water, soy sauce and rock candy with or without antioxidants. The various HAs in marinated food samples and juice were analyzed by HPLC with photodiode-array detection. Results showed that the amount of HAs formed during heating followed an increased order for each increasing heating time. A larger variety and higher amount of HAs were generated in marinated pork when compared to marinated eggs and bean cake. In marinated juice, the levels of HAs were present in greater amount than in marinated foods. The incorporation of antioxidants Vitamin C, Vitamin E and BHT were found to be effective towards HAs inhibition, however, the effect was minor.

Extraction of Cu and Pb from printed circuit board sludge using ammonia solutions.

Sludge from a printed circuit board factory containing high concentrations of Cu and Pb was characterized. Aqueous ammonia solutions were used to extract metals from the sludge. The extraction reactions were completed within 6 hrs. The best extraction efficiency was found at pH of 10.0. Higher solid-liquid ratio and higher ammonia concentration resulted in better extraction efficiency. Fractionation experiments showed that Cu and Pb were mainly extracted from the Fe-Mn oxide-bound and carbonate-bound fractions. Extracted sludge could meet the TCLP regulation limit and be categorized as a non-hazardous waste. Results show that ammonia extraction is of potential in resource recovery and in detoxification of hazardous waste.

Cytotoxicity and antibacterial activity of gold-supported cerium oxide nanoparticles.

BACKGROUND: Cerium oxide nanoparticles (CeO2) have been shown to be a novel therapeutic in many biomedical applications. Gold (Au) nanoparticles have also attracted widespread interest due to their chemical stability and unique optical properties. Thus, decorating Au on CeO2 nanoparticles would have potential for exploitation in the biomedical field. METHODS: In the present work, CeO2 nanoparticles synthesized by a chemical combustion method were supported with 3.5% Au (Au/CeO2) by a deposition-precipitation method. The as-synthesized Au, CeO2, and Au/CeO2 nanoparticles were evaluated for antibacterial activity and cytotoxicity in RAW 264.7 normal cells and A549 lung cancer cells. RESULTS: The as-synthesized nanoparticles were characterized by X-ray diffraction, scanning and transmission electron microscopy, and ultraviolet-visible measurements. The X-ray diffraction study confirmed the formation of cubic fluorite-structured CeO2 nanoparticles with a size of 10 nm. All synthesized nanoparticles were nontoxic towards RAW 264.7 cells at doses of 0-1,000 µM except for Au at >100 µM. For A549 cancer cells, Au/CeO2 had the highest inhibitory effect, followed by both Au and CeO2 which showed a similar effect at 500 and 1,000 µM. Initial binding of nanoparticles occurred through localized positively charged sites in A549 cells as shown by a shift in zeta potential from positive to negative after 24 hours of incubation. A dose-dependent elevation in reactive oxygen species indicated that the pro-oxidant activity of the nanoparticles was responsible for their cytotoxicity towards A549 cells. In addition, cellular uptake seen on transmission electron microscopic images indicated predominant localization of nanoparticles in the cytoplasmic matrix and mitochondrial damage due to oxidative stress. With regard to antibacterial activity, both types of nanoparticles had the strongest inhibitory effect on Bacillus subtilis in monoculture systems, followed by Salmonella enteritidis, Escherichia coli, and Staphylococcus aureus, while, in coculture tests with Lactobacillus plantarum, S. aureus was inhibited to a greater extent than the other bacteria. CONCLUSION: Gold-supported CeO2 nanoparticles may be a potential nanomaterial for in vivo application owing to their biocompatible and antibacterial properties.

Simultaneous determination of prenylflavonoid and hop bitter acid in beer lee by HPLC-DAD-MS.

An HPLC-DAD-MS method with high accuracy and precision was developed for determination of prenylflavonoids and hop bitter acids in beer lee, a by-product from beer brewing process. Four prenylflavonoids and nine hop bitter acids can be simultaneously separated in 29 min using a Thermo HyPURITY C18 column in combination with diode array dectector and mass spectrometer with HPLC solvent gradient system of phosphoric acid aqueous solution at pH 1.6 and acetonitrile at a flow rate of 1.5 mL/min and detection wavelength at 314 nm. Beer lee is found to contain isoxanthohumol (36.2 µg/g), xanthohumol (29.6 µg/g), 8-prenylnaringenin (7.84 µg/g), 6-prenylnaringenin (19.2 µg/g), cohumulone (44.7 µg/g), humulone (123 µg/g), adhumulone (21.8 µg/g), colupulone (44.2 µg/g), lupulone (33.2 µg/g), and adlupulone (5.76 µg/g).

Functional components in Luffa cylindrica and their effects on anti-inflammation of macrophage cells.

The objectives of this study were to determine the distribution of functional components in peel and pulp of Luffa cylindrica and evaluate their anti-inflammatory activity on RAW 264.7 murine macrophage cells. Phenolics and flavonoids were present in abundant amounts in aqueous extract of peel, but in ethyl acetate extracts of peel, oleanolic acid, carotenoids and chlorophylls dominated. Both ethanol and ethyl acetate extracts in peel and pulp decreased production of nitric oxide in LPS-induced RAW 264.7 cells, whereas the ethanol extract mitigated secretion of prostaglandin E(2). Furthermore, all the extracts significantly inhibited IL-6 production, but remained ineffective in retarding generation of IL-1ß and TNF-α. Ethyl acetate extract of peel reduced expression of inducible nitric oxide synthase, but enhanced expression of cyclooxygenase 2. Both ethyl acetate extracts of peel and pulp mitigated expression of p-IκBα, while the latter attenuated expression of p-ERK, and all the extracts failed to inhibit JNK phosphorylation.

Determination of carotenoids in Taraxacum formosanum by HPLC-DAD-APCI-MS and preparation by column chromatography.

The objectives of this study were to determine the variety and content of carotenoids in Taraxacum formosanum, a traditional Chinese herb possessing vital biological activities, by developing an HPLC-DAD-APCI-MS method and a preparative column chromatographic method for carotenoid isolation. A total of 25 carotenoids were resolved within 66 min by employing a YMC C30 column and a gradient mobile phase of methanol-acetonitrile-water (79:14:7, v/v/v) and methylene chloride (100%) with flow rate at 1.0 mL/min and detection at 450 nm. All-trans-canthaxanthin was shown to be an appropriate internal standard for quantitation, with all-trans-ß-carotene and its cis isomers present in largest amount (413.6 µg/g), followed by all-trans-violoxanthin and its cis isomers (209.5 µg/g), all-trans-lutein and its cis isomers (212.4 µg/g), all-trans-neoxanthin and its cis isomers (134.6 µg/g), antheraxanthin (16.5 µg/g), all-trans-ß-cryptoxanthin and its cis isomers (5.8 µg/g), all-trans-zeaxanthin (3.6 µg/g) and neochrome (0.1 µg/g). For preparative chromatography, with a glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) as adsorbent, the carotenoid fraction was eluted with 300 mL of ethyl acetate with flow rate at 10 mL/min. Some more epoxides and cis isomers of carotenoids were generated during preparative column chromatography. Nevertheless, the carotenoids isolated from T. formosanum may be used as raw material for possible production of health food in the future.

Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC-DAD-ESI-MS.

A high-performance liquid chromatography-diode array detection-mass spectrometry method with electrospray ionization mode (HPLC-DAD-ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC-DAD-ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile-water (94:6, v/v) with flow rate at 1mL/min, column temperature at 30 degrees C and detection wavelength at 280nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6microg/g), followed by quercetin-3-O-rutinoside (281.3microg/g), dicaffeoylquinic acid isomers (250.1microg/g), chlorogenic acid (237.0microg/g), quercetin-di-(rhamnohexoside) (117.5microg/g), quercetin-di-(rhamno)-hexoside (116.8mug/g), kaempferol-3-O-rutinoside (97.7microg/g), isorhamnetin-3-O-rutinoside (72.1microg/g), p-coumaric acid (64.0microg/g), caffeic acid (23.7microg/g) and vanillic acid (22.8microg/g).

Inhibition effect of poly(γ-glutamic acid) on lead-induced toxicity in mice.

The objectives of this study were to evaluate the efficiency in treatment of lead-induced intoxication in mice with γ-PGA as chelating agent and compare with the drug (meso-2,3-dimercaptosuccinic acid). The results showed the incorporation of γ-PGA at 200 and 400 mg/kg could reduce the accumulation of lead in the liver, heart, and testis; however, the latter was more effective in decreasing the lead content in the kidney and spleen. Nevertheless, both doses failed to inhibit the lead accumulation in the lung and brain. Additionally, both doses of γ-PGA could reduce TBARs in the kidney and brain, as well as elevate δ-aminolevulinic acid dehydrase (δ-ALAD) activity in blood and decrease glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH) activities in the serum. For hematological parameters, both white blood cells (WBCs) and hematocrite (HCT) were raised by 400 mg/kg of γ-PGA, while for both doses of γ-PGA, a slight decline in hemoglobin (HGB), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) was observed, with the red blood cells (RBCs) being unaffected.

Determination of flavonoids and saponins in Gynostemma pentaphyllum (Thunb.) Makino by liquid chromatography-mass spectrometry.

Gynostemma pentaphyllum (Thunb.) Makino, a traditional Chinese herb possessing antitumor and antioxidant activities, has been shown to contain several functional components like saponins and flavonoids. However, their identities remain uncertain. The objectives of this study were to develop an appropriate extraction, purification and HPLC-MS method to determine saponins and flavonoids in G. pentaphyllum. Both flavonoids and saponins were extracted with methanol, followed by purification with a C18 cartridge to elute the former with 50% methanol and the latter with 100% methanol. A total of 34 saponins were separated within 40 min by a Gemini C18 column and a gradient mobile phase of acetonitrile and 0.1% formic acid in water, in which 18 saponins were identified by LC-MS with ESI mode and Q-TOF (LC/MS/MS). Similarly, a total of eight flavonoids were separated within 45 min by the same column and a gradient solvent system of methanol and 0.1% formic acid in water, with identification being carried out by a post-column derivatization method and LC-MS with ESI mode. The amounts of flavonoids in G. pentaphyllum ranged from 170.7 to 2416.5 mug g(-1), whereas saponins were from 491.0 to 89,888.9 mug g(-1).

A proton-coupled conformational switch of Escherichia coli 5S ribosomal RNA.

We report temperature-jump kinetic studies of the early melting transition of Escherichia coli 5S rRNA. A single measurable relaxation time tau, independent of concentration, was found at 266 nm. We monitored the transition temperature tm for this process (in the range from 0 to 40 degrees C) as a function of Mg2+, Na+, K+, spermidine, and H+ concentrations. Contrary to the usual effect of salts on nucleic acid stability, addition of mono- and multivalent counterions decreases tm for the early melting transition. Also unexpectedly, we found a strong dependence of tm on pH in the physiological range of 7--8. Quantitative analysis of the data indicates that about 0.7 protons are release when the ordered (low-temperature) form melts, whereas about 2 NA+ (or K+) and 0.5 Mg2+ are taken up by the melted (high-temperature) form. We estimate the enthalpy of the transition to be 15--20 kcal/mol (63--84 kJ/mol) and also report the forward and reverse rate constants and activation energies for the transition, along with the influence of ions on the transition dynamics. Diffusion constant measurements reveal that the low-temperature form has a frictional coefficient about 10% larger than that of the high-temperature form. The data imply a low-temperature tertiary structure capable of binding a proton. Increase of pH, temperature, or counterion concentration (all at near-physiological values) causes a tertiary conformational switch to a more compact form that has greater counterion binding but less proton binding. We discuss possible physiological roles for the transition.

Expression of two S-ribonucleases of Petunia inflata using baculovirus expression system.

We have previously shown that three Petunia inflata S-proteins, products of the multiallelic S-gene of the self-incompatibility system, are ribonucleases. Here we report the expression of cDNAs for two of these S-proteins using the baculovirus expression system. S2- and S3-proteins were found in both supernatants and lysates of Spodoptera frugiperda cells infected with recombinant baculoviruses. Both recombinant S-proteins contained glycosylated (25 kD) and nonglycosylated (23 kD) forms. Recombinant S2- and S3-proteins were purified from insect cell cultures, and the amino-terminal sequences determined from glycosylated S2- and S3-proteins indicated that the leader peptide encoded by each cDNA was correctly removed. Both glycosylated and nonglycosylated forms of S2- and S3-proteins exhibited ribonuclease activity.

Excess nonsynonymous substitution of shared polymorphic sites among self-incompatibility alleles of Solanaceae.

The function of the self-incompatibility locus (S locus) of many plant species dictates that natural selection will favor high levels of protein diversity. Pairwise sequence comparisons between S alleles from four species of Solanaceae reveal remarkably high sequence diversity and evidence for shared polymorphism. The level of amino acid constraint was found to be significantly heterogeneous among different regions of the gene, with some regions being highly constrained and others appearing to be virtually unconstrained. In some regions of the protein, there was an excess of nonsynonymous over synonymous substitution, consistent with the strong diversifying selection that must operate on this locus. These hypervariable regions are candidates for the sites that determine functional allelic identity. Simple contingency table tests show that sites that have polymorphism shared between species have more nonsynonymous substitution than polymorphic sites that do not exhibit shared polymorphism. This is consistent with the idea that adaptive evolution favoring amino acid replacement is occurring at sites with shared polymorphism. Tests of clustered polymorphism reveal that an unusually low rate of recombination must be occurring in this locus, allowing very ancient alleles to preserve their identity.

How flowering plants discriminate between self and non-self pollen to prevent inbreeding.

Flowering plants have evolved various genetic mechanisms to circumvent the tendency for self-fertilization created by the close proximity of male and female reproductive organs in a bisexual flower. One such mechanism is gametophytic self-incompatibility, which allows the female reproductive organ, the pistil, to distinguish between self pollen and non-self pollen; self pollen is rejected, whereas non-self pollen is accepted for fertilization. The Solanaceae family has been used as a model to study the molecular and biochemical basis of self/non-self-recognition and self-rejection. Discrimination of self and non-self pollen by the pistil is controlled by a single polymorphic locus, the S locus. The protein products of S alleles in the pistil, S proteins, were initially identified based on their cosegregation with S alleles. S proteins have recently been shown to indeed control the ability of the pistil to recognize and reject self pollen. S proteins are also RNases, and the RNase activity has been shown to be essential for rejection of self pollen, suggesting that the biochemical mechanism of self-rejection involves the cytotoxic action of the RNase activity. S proteins contain various numbers of N-linked glycans, but the carbohydrate moiety has been shown not to be required for the function of S proteins, suggesting that the S allele specificity determinant of S proteins lies in the amino acid sequence. The male component in self-incompatibility interactions, the pollen S gene, has not yet been identified. The possible nature of the pollen S gene product and the possible mechanism by which allele-specific rejection of pollen is accomplished are discussed.

S-alleles are retained and expressed in a self-compatible cultivar of Petunia hybrida.

We identified two S-allele-associated proteins (S-proteins) in a self-compatible cultivar of Petunia hybrida based on their segregation in F1 hybrids between P. hybrida and its self-incompatible relative, Petunia inflata (with S2S2 genotype), and in selfed progeny of P. hybrida. These two S-proteins, designated Sx-protein (24 kDa) and So-protein (31 kDa), are pistil specific, and their expression follows a temporal and spatial pattern similar to that of S-proteins characterized in self-incompatible solanaceous species. Their amino-terminal sequences also share a high degree of similarity with those of solanaceous S-proteins. Selfing of P. hybrida yielded plants with SoSo,SxSo, and SxSx genotypes in an approximately 1:2:1 ratio, indicating that the Sx-and So-alleles, though expressed in the pistil, failed to elicit a self-incompatibility response. The S2-allele of P. inflata is expressed in all the F1 hybrids, rendering them capable of rejecting pollen bearing the S2-allele. The So-allele is not functional in the F1 hybrids, because all the F1 progeny with S2So genotype are self-compatible. However, in F1 hybrids with S2Sx genotype, approximately half are self-incompatible and half are self-compatible, indicating that the function of the Sx-allele depends on the genetic background. These results strongly suggest that the presence of functional S-alleles alone is not sufficient for expression of a self-incompatibility phenotype, and reaffirm the multigenic nature of gametophytic self-incompatibility suggested by earlier genetic studies.