Evaluation of Appropriate Conditions for Efficient Simultaneous Determination of US EPA and EU Priority Polycyclic Aromatic Hydrocarbons in Various Food Categories and Assessment of Their Consumption Risk.

The QuEChERS (quick, easy, cheap, effective, rugged and safe) conditions were optimized for efficient determination of the U.S. Environmental Protection Agency (US EPA) and European Union (EU) priority polycyclic aromatic hydrocarbons (PAHs) for the categories of grains, tuber & starchy vegetables, soy beans and products, fish & seafood, and poultry & meat, including raw materials and their corresponding products. The PAHs were analyzed using ultrahigh-performance liquid chromatography with temperature-controlled fluorescence detection and gas chromatography with tandem mass spectrometry. The established conditions had good accuracy, repeatability, and precision. Environmental pollution and processing methods influence the level of PAHs in samples. The low molecular weight PAHs were present in all raw materials, and processing increased high and low molecular weight PAHs in the products. The excess cancer risk for consumption of PAHs in cooked samples was mostly acceptable; a small number of samples might be of slight concern in certain age groups.

Establishment of optimal QuEChERS conditions of various food matrices for rapid measurement of heterocyclic amines in various foods.

The optimal conditions for QuEChERS (quick, easy, cheap, effective, rugged and safe) with superior performance were established to rapidly extract 21 heterocyclic amines (HAs) from 9 categories of food matrices including meat and poultry, eggs, soy beans and products, composite foods, fish and seafood, grains, beer, dairy foods, and coffee. The QuEChERS conditions and the developed ultra-high performance liquid chromatography-tandem mass spectrometry analysis conditions were then applied to the determination of HAs in popular food products sold in the Taiwan market. The conditions comply with the food chemical analysis specifications of Taiwan Food and Drug Administration. Coffee products and braised products that require a longer cooking time contained relatively high content of HAs (mainly Harman and Norharman), and their consumption is relatively high resulting in relatively high intake of HAs from these products. The dietary intake of HAs in plant-based protein food products should also be of concern.

Establishment of an appropriate method for determining multiple heterocyclic amines in soy products processed with various methods.

A method using UPLC-MS/MS and a core-shell C18 column was developed to simultaneously determine 21 heterocyclic amines (HAs) in 15 min. Appropriate QuEChERS conditions were also established to conveniently extract HAs from soy products cooked with various methods. These conditions presented good analytical performance; limit of detection, limit of quantification, recovery (%), repeatability (coefficient of variation (CV) %) and intermediate precision (CV%) were 0.008 âˆ¼ 0.150 ng/g, 0.025 âˆ¼ 0.500 ng/g, 62 âˆ¼ 91%, ≤ 28% and ≤ 23% for tofu sample, and 0.003 âˆ¼ 0.100 ng/g, 0.010 âˆ¼ 0.350 ng/g, 64 âˆ¼ 93%, ≤ 19% and ≤ 20% for soy milk sample, respectively. HAs contents in the samples increased with cooking temperature and time. The tofu samples cooked by frying had much higher HAs content than those cooked by boiling and roasting. Norharman and Harman mainly contributed HAs content in all samples. For the general population in Taiwan, the highest estimated level of HAs consumed from the samples is 373.67 ng/day.

Litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction exhibited protective efficacy to suppress nickel-induced expression for vascular endothelial growth factor in HepG2 cells.

The protective efficacy of litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction (LFPF) composed of (-)-epicatechin and proanthocyanidin A2 against vascular endothelial growth factor (VEGF) generation induced by nickel (Ni) in hepatocellular carcinoma (Hep G2) cells was studied. VEGF is an angiogenic inducer, which promotes tumor angiogenesis, leading to rapid tumor growth and metastasis. VEGF could be substantially induced in the Ni-mediated Hep G2 cells. Through LFPF treatment, the Ni-induced VEGF generation could be suppressed significantly. The inhibition of HIF-1α expression by blocking phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways, and the suppression of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT 3), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)/mitogen-activated protein kinase (MEK1/2)/extracellular-signal-regulated kinase (ERK1/2) pathways are important molecular mechanisms for the LFPF action. LFPF should probably reduce the risk of liver cancer in Ni-contaminated environments by inhibiting VEGF expression. PRACTICAL APPLICATIONS: LFPF mainly contained (-)-epicatechin and proanthocyanidin A2. Our results demonstrated that LFPF considerably suppressed the Ni-induced VEGF expression through inhibition of JAK2/STAT 3 and RAF1/MEK1/2/ERK1/2 pathways and prohibited HIF-1α expression through blocking PI3K/AKT/mTOR pathway. Litchi flowers might have the potential to diminish the liver cancer risk in a Ni-contaminated environment through suitable treatment.

Phenolic compositions and antioxidant properties of leaves of eight persimmon varieties harvested in different periods.

The compositions and contents of antioxidant components and antioxidant attributes (scavenging DPPH radicals, TEAC, ferric reducing power and inhibiting Cu2+-induced human LDL oxidation) for the leaves of eight persimmon varieties harvested from Sep. to Nov. were determined. Harvest time and variety were important factors affecting the compositions and contents of phenolic compounds in persimmon leaves; moreover, phenolic contents (polyphenol, flavonoid, condensed tannin and phenolic acid) of the leaves were significantly correlated with their antioxidant activities. For each variety, the leaves harvested in months with higher temperature, solar radiation and sunshine duration had higher phenolic contents contributing to better antioxidant properties (ranking: Sep. > Oct. > Nov.). In addition, the compositions and contents of phenolic components and antioxidant capacities for the leaves from various persimmon varieties were also different. The leaves of persimmon varieties belonging to pollination constant and astringent (PCA) had higher phenolic contents and also presented better antioxidant effects.

Composition of phenolic compounds and antioxidant attributes of Cyclea gracillima Diels extracts.

Cyclea gracillima Diels is a Taiwanese native medicinal herb. However, there are currently few relevant reports on its biochemical activity. In this study, the antioxidant attributes of the ethanol and hot water extracts of this herb were assayed using in vitro models, including the following: 2,2-diphenyl-1-(2,4,6-trinitrophenyl)-hydrazyl radical scavenging, Trolox equivalent antioxidant capacity, reducing power, and chelating ferrous ions. The following biochemical models were also assayed: inhibition of human low density lipoprotein oxidation, inhibition of human erythrocyte hemolysis, and scavenging oxygen radicals in human blood. The composition and content of flavonoids and phenolic acids in these extracts were also analyzed. The results showed that these extracts with high polyphenol levels presented remarkable antioxidant effects in all assays, especially when extracted with ethanol. Six phenolic acids (mainly ferulic acid, sinapic acid, and syringic acid) and 12 flavonoids (mainly narigenin, myricetin, naringin, and apigenin) were found in these extracts.

Effective compounds in the fruit of Muntingia calabura Linn. cultivated in Taiwan evaluated with scavenging free radicals and suppressing LDL oxidation.

Scavenging effect of 2,2-diphenyl -2-picrylhydrazyl hydrate (DPPH) radicals, inhibitory effect of low-density lipoprotein (LDL) oxidation, Trolox equivalent antioxidant capacity (TEAC), and phenolic contents were used for the activity-guided separation to identify the effective compounds of Muntingia calabura Linn. fruit. Its ethanol extract with higher phenolic content and antioxidant activities was subjected to silica gel column chromatographic separation, which was sequentially eluted with n-hexane, 10-90% ethyl acetate (EA) in n-hexane, EA, EA/acetone (50/50, v/v), acetone, acetone/methanol (MeOH) (50/50, v/v), and MeOH; fifteen fractions (Fr. 1-15) were obtained. Fractions 13 and 14 with better antioxidant effects were mixed followed by purification of the effective compounds using HPLC. Two major compounds were isolated and identified as gallic acid and 1,2-benzenedicarboxylic acid diisooctyl ester through high performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) measurements. Their amounts in the fruit were 3.76 and 4.62 mg g-1. This study is the first report to clarify the effective antioxidant compounds of M. calabura Linn. fruit.

Molecular mechanisms of the effects of the ethanolic extract of Muntingia calabura Linn. fruit on lipopolysaccharide-induced pro-inflammatory mediators in macrophages.

Four flavonoids (epicatechin, rutin, diosmin and luteolin) and 11 phenolic acids (gallic acid, gentisic acid, p-hydroxybezoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, syringic acid, p-anisic acid and rosmarinic acid) were determined in the ethanolic extract of M. calabura Linn. fruit gathered in Taiwan. The extract suppressed the lipopolysaccharide-stimulated expressions of inducible nitric oxide synthase and cyclooxygenase-2 as well as the productions of nitric oxide, prostaglandin E2 and pro-inflammatory cytokines [tumour necrosis factor-α, interleukin (IL)-1ß and IL-6] in RAW264.7 macrophages. The extract modulated the inflammatory processes through inactivation of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs) p38 and c-Jun NH2-terminal kinase 1/2 (JNK1/2), and Janus kinase 2 (JAK2)/signal transducers and activators of transcription 1/3 (STAT1/3). Moreover, the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) followed by inducing the production of heme oxygenase-1 (HO-1) is also related to the anti-inflammatory effect of the extract.

Phenolic compositions and antioxidant attributes of leaves and stems from three inbred varieties of Lycium chinense Miller harvested at various times.

Antioxidant components and properties (assayed by scavenging DPPH radicals, TEAC, reducing power, and inhibiting Cu(2+)-induced human LDL oxidation) of leaves and stems from three inbred varieties of Lycium chinense Miller, namely ML01, ML02 and ML02-TY, harvested from January to April were studied. Their flavonoid and phenolic acid compositions were also analyzed by HPLC. For each variety, the leaves and stems collected in higher temperature month had higher contents of total phenol, total flavonoid and condensed tannin. Contents of these components in the samples collected in different months were in the order: April (22.3°C)>March (18.0°C)>January (15.6°C)>February (15.4°C). Antioxidant activities of the leaves and stems for all assays also showed similar trends. The samples from different varieties collected in the same month also possessed different phenolic compositions and contents and antioxidant activities. Their antioxidant activities were significantly correlated with flavonoid and phenolic contents.

Effects of roasting temperature and duration on fatty acid composition, phenolic composition, Maillard reaction degree and antioxidant attribute of almond (Prunus dulcis) kernel.

Roasting treatment increased levels of unsaturated fatty acids (linoleic, oleic and elaidic acids) as well as saturated fatty acids (palmitic and stearic acids) in almond (Prunus dulcis) kernel oils with temperature (150 or 180 °C) and duration (5, 10 or 20 min). Nonetheless, higher temperature (200 °C) and longer duration (10 or 20 min) roasting might result in breakdown of fatty acids especially for unsaturated fatty acids. Phenolic components (total phenols, flavonoids, condensed tannins and phenolic acids) of almond kernels substantially lost in the initial phase; afterward these components gradually increased with roasting temperature and duration. Similar results also observed for their antioxidant activities (scavenging DPPH and ABTS(+) radicals and ferric reducing power). The changes of phenolic acid and flavonoid compositions were also determined by HPLC. Maillard reaction products (estimated with non-enzymatic browning index) also increased with roasting temperature and duration; they might also contribute to enhancing the antioxidant attributes.

Inhibitory effect of litchi (Litchi chinensis Sonn.) flower on lipopolysaccharide-induced expression of proinflammatory mediators in RAW264.7 cells through NF-κB, ERK, and JAK2/STAT3 inactivation.

Litchi (Litchi chinensis Sonn.) flower ethanolic extract (LFEE) was found to contain five flavanoids [total amount, 102.73 ± 5.50 mg/g of dried extract (gDE)], nine phenolic acids (total amount, 60.31 ± 4.52 mg/gDE), and proanthocyanidin A2 (79.31 ± 2.95 mg/gDE). LFEE was used to evaluate the inhibitory effects on lipopolysaccharide- (LPS-) induced pro-inflammatory mediators in RAW264.7 cells. The results showed that LFEE treatment could suppress the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the productions of nitric oxide (NO) and prostaglandin E2 (PGE2), and the secretions of pro-inflammatory cytokines [interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α)] in the LPS-mediated RAW264.7 cells. The attenuation of LPS-induced inflammatory responses by LFEE was found to be closely related to the inhibition of the translocation of nuclear factor κB (NF-κB) p50/p65 subunits correlated with suppression of the activation of the inhibitor of κB kinase (IKK) α/ß and downregulation of activation of extracellular signal-regulated kinase (ERK) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3).

Antioxidant effect and active components of litchi (Litchi chinensis Sonn.) flower.

The effects of scavenging 2, 2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radicals and inhibiting low-density lipoprotein (LDL) oxidation, and phenolic quantities were used for the activity-guided separation to identify the effective components of litchi flower. The acetone extract of the flower with notable antioxidant capacities was suspended in water and sequentially partitioned with n-hexane, ethyl acetate (EA) and n-butanol. The EA partition with the highest phenolic levels and antioxidant capacities was subjected to silica gel column chromatography. Thirteen fractions (Fr. 1-13) were collected; Fr. 10-12 with higher phenolic levels and antioxidant effects were applied to Sephadex LH-20 column chromatography. Each fraction was further separated into three sub-fractions and the second ones (Fr. 10-II, 11-II, and 12-II) were the best, which two major compounds could be isolated by semi-preparative high performance liquid chromatography (HPLC). Through Mass (MS) and Nuclear Magnetic Resonance (NMR) measurements, they could be identified as (-)-epicatechin and proanthocyanidin A2. Their contents in the litchi flower were 5.52 and 11.12 mg/g of dry weight, respectively. The study was the first time to reveal the effective antioxidant components of litchi flower.

Simultaneous Determination of Furostanol, Pennogenyl, and Diosgenyl Glycosides in Taiwanese Rhizoma Paridis ( Paris formosana Hayata) by high-performance liquid chromatography with evaporative light scattering detection.

A high-performance liquid chromatographic method with an evaporative light scattering detector (HPLC-ELSD) was developed to simultaneously determine 10 steroidal saponins, including 3 furostanol glycosides, 3 pennogenyl glycosides, and 4 diosgenyl glycosides in Taiwanese rhizoma paridis ( Paris formosana Hayata). The condition was a Cosmosil C18 column kept at 35 °C and a step-gradient solvent system consisting of acetonitrile and water (25:75, v/v) in the first 30 min, 45:55 (v/v) from 31 to 45 min, and 50:50 (v/v) from 45 to 65 min, at a flow rate of 1 mL/min. The separation factors (α) and resolutions (Rs) were better than 1, and the limits of detection (LODs) and limits of quantification (LOQs) were 0.01-0.27 and 0.04-0.90 µg, respectively, for these saponins. Moreover, 203 nm UV detection was also used for comparison. The saponins in P. formosana Hayata gathered from various areas of Taiwan were determined by applying the established method.

Determination of carotenoids in Dunaliella salina cultivated in Taiwan and antioxidant capacity of the algal carotenoid extract.

A simple HPLC method with good separation efficiency was developed to determine all-trans and cis forms of carotenoids in Dunaliella salina cultivated in Taiwan. The analysis used a C30 column (250×4.6mm, 5µm) and an isocratic solvent system (flow rate=1mL/min) mixing methanol-acetonitrile-water (84/14/2, v/v/v) and methylene chloride, (75/25, v/v). Carotenoids were detected at 450nm. Moreover, the antioxidant capacities of the algal carotenoid extract were also evaluated with Trolox equivalent antioxidant capacity (TEAC) assay, reducing power and 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging assay. Results showed that 7 carotenoids in the algal extract could be separated simultaneously within 30min and the total amount of them was 290.77mg/g algae. The contents of all-trans-ß-carotene and 9- or 9'-cis-ß-carotene, the major carotenoids in the algae, were 138.25 and 124.65mg/g algae, respectively. The contents of all-trans-lutein, all-trans-zeaxanthin, 13- or 13'-cis-ß-carotene, all-trans-α-carotene and 9- or 9'-cis-α-carotene were 6.55, 11.27, 4.95, 2.69, and 2.41mg/g algae, respectively. The algal carotenoid extract had significantly higher antioxidant activity than all-trans forms of α-carotene, ß-carotene, lutein and zeaxanthin in all antioxidant assays. The cis forms of carotenoids, especially 9- or 9'-cis-ß-carotene, might play crucial roles for the antioxidant capacities of the algal extract.

Determination of steroidal saponins in different organs of yam (Dioscorea pseudojaponica Yamamoto).

Yams (Dioscorea spp.) are perennial trailing rhizome plants. Steroidal saponins, furostanol and spirostanol glycosides are the marked functional compounds in yams. In this investigation, a C18 solid phase extraction method was developed for yam saponins purification. The contents of saponins in various organs of yam (Dioscorea pseudojaponica Yamamoto) were also determined. Results showed that the recoveries of yam saponins extracted by the developed method were about 99.48-100.08% when the saponins (each saponin weighed 0.20, 0.50 and 1.00mg) passing through the C18 cartridge. The extractive method could efficiently reduce the interferences from impurities in yam saponin extracts prior to HPLC analysis. The recoveries of added saponins in different yam organs were 98.34-99.92% for tuber flesh, 95.98-98.89% for tuber cortex, 97.89-99.44% for rhizophor, 93.82-98.01% for leaf and 93.87-97.65% for vine, respectively. The yam tuber cortex had the highest amount of saponins (582.53µg/gdw), which was higher than that existed in the tuber flesh (227.86µg/gdw) about 2.55 times. The contents of saponins in the rhizophor, leaf and vine of yam were 29.39, 24.41 and 23.96µg/gdw, respectively.

Effects of different steeping methods and storage on caffeine, catechins and gallic acid in bag tea infusions.

Bag teas, packed 3g of ground black, green, oolong, paochoung and pu-erh tea leaves (the particle size used was 1-2mm), were steeped in 150 mL of 70, 85 or 100 degrees C hot water to study the effects of the number of steeping (the same bag tea was steeped repeatedly eight times, 30s each time, as done in China for making ceremonial tea) and varied steeping durations (0.5-4 min) on caffeine, catechins and gallic acid in tea infusions. The changes in tea infusions during storage at 4 or 25 degrees C for 0-48 h and the variations in these compounds of bag tea infused with 150 mL of 4 or 25 degrees C cold water for 0.5-16 h were also investigated. A HPLC method with a C18 column and a step gradient solvent system consisting of acetonitrile and 0.9% acetic acid in deionized water was used for analysis. Results for all kinds of tea samples showed that the second tea infusion contained the highest contents of caffeine, catechins and gallic acid when bag teas were steeped in 70 degrees C water. It was different from that steeped at 85 and 100 degrees C, the highest contents existed in the first infusion. These compounds decreased gradually in later infusions. Higher amounts of caffeine, catechins and gallic acid could be released from bag teas as hotter water was used. As steeping duration prolonged, these ingredients increased progressively, however, their levels were lower than that cumulated from the infusions with the identical bag tea prepared recurrently at the same temperature and time points. (-)-Gallocatechin gallate and (+)-catechin existed in these tea infusions rarely and could not be detected until a certain amount of them infusing. Except gallic acid that showed a significant increase and caffeine that exhibited no significant change, all kinds of catechins decreased appreciably after tea infusions were stored at 25 degrees C for 36 h; nevertheless, all of them showed no evident changes at 4 degrees C storage. The caffeine, catechins and gallic acid in tea infused with cold water also increased with increasing duration. Their contents in 25 degrees C steeped tea were higher than that made at 4 degrees C; moreover, their infusion rates from bag teas to cold water were markedly lower than that steeped in hot water. Infusing efficiencies of non-gallated catechins were higher than gallated catechins under cold water steeping.

Effects of domestic processing on steroidal saponins in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto).

The effects of domestic processing on steroidal saponins and furostanol and spirostanol glycosides in Taiwanese yam cultivar (Dioscorea pseudojaponica Yamamoto) were studied. The baking or frying of yam slices was conducted at 150, 180, and 200 degrees C for 3, 5, and 10 min. Yam slices were steamed or microwave cooked at 2450 MHz with an output power of 850 W for 3, 5, and 10 min. The various saponins were quantified by HPLC with an evaporative light scattering detector (ELSD). Results showed that the contents of saponins were decreased along with increasing cooking temperature and time except for the steaming treatment. None of the steamed yam slices significantly change their initial compositions or quantities of furostanol and spirostanol glycosides. Fried yam slices had the highest loss of saponins, especially at 200 degrees C for 10 min (93 and 97% reductions for total furostanol and spirostanol glycosides, respectively). After baking for 10 min at 200 degrees C, the total furostanol and spirostanol glycosides were reduced by 67 and 74%, respectively. There were 12, 44, and 84% decreases for total furostanol glycosides and 10, 35, and 75% reductions for total spirostanol glycosides in yam slices after microwave cooking for 3, 5, and 10 min, respectively. Diosgenin, the aglycone of these saponins, could be found in yams after microwave cooking and baking, but not in steamed and fried yams.