Multiple-Reaction Monitoring Tandem Mass Method for Determination of Phenolics and Water-Soluble Vitamins in Eccoilopus formosanus.

This study established a validated method for the quantitative and qualitative determination of eight signature compounds in Eccoilopus formosanus. We used multiple-reaction monitoring scanning for quantification, and switched the electrospray ion source polarity between positive and negative modes in a single chromatographic run. The precursor-to-product ion transitions were m/z 355/163, m/z 181/163, m/z 265/122, m/z 269/117, m/z 170/152, m/z 377.2/180.7, m/z 169/124.8 and m/z 193/134 for chlorogenic acid, caffeic acid, thiamine, apigenin, pyridoxamin, riboflavin, gallic acid and ferulic acid, respectively. The developed method was also validated for accuracy, precision and limit of quantification. In this method, eight compounds were quantified with correlation coefficients of greater than 0.995. A high recovery (81.5-94.1%) and good reproducibility was obtained for five phenolics and three vitamins with the relative standard deviation, ranging from 1.2 to 3.5%. This method may be applied to the determination of both phenolics and water-soluble vitamins in cereal grain. The results may suggest that the extract of E. formosanus could be a good source of bioactive phytochemicals.

HPLC-DAD-ESI-MS Analysis for Simultaneous Quantitation of Phenolics in Taiwan Elderberry and Its Anti-Glycation Activity.

Sambucus formosana is most commonly used as a traditional herb medicine in Taiwan. In this study, high performance liquid chromatography equipped with photodiode array detection-mass (HPLC-DAD-ESI-MS) method was developed for the identification and quantification of bioactive phenolics. The developed method was also validated for accuracy, precision, limit of detection, and quantification. In this method, chlorogenic acid, rutin, isoquercetrin, nictoflorin, astragalin, and quercetin were quantified in linearity range of 10-100 (µg/mL) with a correlation coefficient of greater than 0.996. High recovery (86.5-93.1%) and good reproducibility were obtained for six phenolics with the relative standard deviation ranging from 1.7-3.1%. Therefore, the proposed method for simultaneous quantification of six bioactive phenolics in the extract and fractions of S. formosana using HPLC-DAD-ESI-MS detection under the optimized conditions is accurate and validated. Among the results, methanol extract showed the greatest values of total phenolic content (93.1 mg gallic acid equivalent/g). Additionally, the methanol extract revealed best antioxidant capacity based on the DPPH scavenging activity and anti-glycation activity (IC50 was observed at 97.1 and 77.9 µg/mL, respectively).

Extraction Optimization of Flavonoids from Hypericum formosanum and Matrix Metalloproteinase-1 Inhibitory Activity.

Hypericum formosanum is a valuable herb in Taiwan. In this study, response surface methodology was employed to optimize the ultrasound-assisted extraction of flavonoids from Hypericum formosanum. A central composite design with three variables (ethanol concentration, extraction time, and extraction temperature) was applied. Experimental results were fitted to the second order polynomial model and one-way analysis of variance was used to determine the goodness of fit of the model and the optimal conditions for responses. The optimal conditions for the maximum extraction yield of total flavonoid content (101.1 mg/g) using ultrasound-assisted extraction were ethanol concentration, 73.5%; extraction time, 38.3 min; and extraction temperature, 62.5 °C. The predicted result was consistent with the experimental result obtained under optimal extraction conditions. Hyperoside, astilbin, quercitrin, and quercetin from Hypericum formosanum extract (HFE) were identified by Ultra performance liquid chromatography-diode array detector-mass (UPLC-DAD-MS). HFE significantly reduced matrix metalloproteinase-1 protein expression in human skin keratinocyte cells, induced by advanced glycation end products.

The effect of papain hydrolysis on tropomyosin levels in shrimp.

Shrimp allergy is a serious public health problem. Epidemiological studies indicated the main cause of food allergy is the ingestion of shrimp and crab. Consequently, reducing the risk of shrimp and crab allergy is a major research focus. Few studies have demonstrated the effect of enzyme hydrolysis on reduction of shrimp allergens in food. In this study, we used papain, a commonly used enzyme in the food industry, as an ingredient to decrease levels of allergen tropomyosin in shrimp. 0-50U of papain was used to treat the shrimp meat, and then heated to measure the levels of tropomyosin, and determined the change of the protein secondary structure. The results showed that 20U of papain decrease the tropomyosin levels up to 80 %, which was further enhanced when combined with 3 min of heating. Furthermore, the result of Fourier-transform infrared spectroscopy (FTIR) showed alteration of secondary protein structure after the processing. The processing developed in this study may be an effective method used to change the levels and structure of tropomyosin.

Antioxidant Activity and Inhibitory Effects of Black Rice Leaf on the Proliferation of Human Carcinoma Cells.

The leaves of black rice, well-known as postharvest agricultural waste, contain a rich source of antioxidants with multiple benefits for human health. In the present study, the ethyl acetate fraction obtained from black rice leaf was separated into five subfractions using Sephadex LH-20 column chromatography, and their antioxidant and anticancer activities were investigated. The results revealed that among all the subfractions, subfraction 5 (Sub5) showed the highest total phenolic and flavonoid values. The antioxidant activity was also superior in Sub5 (the IC50 values are 3.23, 31.95, and 72.74 µg/mL, in the DPPH, ABTS, and reducing power assays, respectively) compared to the other subfractions. All subfractions, in a time-dependent manner, inhibited the proliferation of hepatoma (HepG2), breast (MCF-7), and colorectal (Caco-2) cancer cells, especially the Sub5. Thus, Sub5 was employed to conduct the cell cycle and cell apoptosis by flow cytometry. Sub5 significantly increased the accumulation of cells at the Sub-G1 phase in HepG2 cells (44.5%, at 48 h). Furthermore, it could trigger annexin V-detected apoptosis through mitochondrial and death receptor pathways accompanied by the suppression of PI3K/Akt and Erk signaling pathways. In addition, HPLC-DAD-MS/MS was conducted to characterize the bioactive constituents in the most potent antioxidant, cytotoxic, and apoptosis-inducing subfraction. Conclusively, Sub5 may have high potential as functional dietary supplements to inhibit the development of HepG2 liver cancer.

Evaluation of Phytochemical Contents and In Vitro Antioxidant, Anti-Inflammatory, and Anticancer Activities of Black Rice Leaf (Oryza sativa L.) Extract and Its Fractions.

Black rice leaves (Oryza sativa L.) are a major part of rice straw left in open fields after rice harvest as agricultural waste. In this study, crude ethanolic extract (CEE) and various solvent fractions (hexane (Hex), ethyl acetate (EtOAc), n-butanol (n-BuOH), and aqueous fractions) of black rice leaves were investigated for their bioactive compound contents as well as antioxidant, anti-inflammatory, and anticancer activities. The results demonstrated that among all the fractions, the n-BuOH fraction presented the greatest contents of total phenolics and flavonoids, while anthocyanins were found to be abundant in the n-BuOH and aqueous fractions, which also exhibited powerful antioxidant abilities according to DPPH and ABTS radical-scavenging assays and a reducing power assay. Regarding anti-inflammatory activity, CEE and EtOAc reduced the production of NO and cytokine secretion (PGE2, IL-6, and IL-1ß) but displayed less effect on tumor necrosis factor α (TNF-α) release in lipopolysaccharide (LPS)-induced RAW 264.7 cells. They also significantly decreased iNOS and COX-2 protein expression. Additionally, the phenolics-rich ethyl acetate fraction showed the greatest activity against HepG2 liver carcinoma cells, inhibited cell growth, increased the Sub-G1 population, and induced apoptosis via mitochondrion-dependent mechanisms. In conclusion, black rice leaves, a byproduct of rice, exhibited strong antioxidant, anti-inflammatory, and anticancer capacities and might be useful for application in functional foods and the pharmaceutical industry.

Development and Optimization of Djulis Sourdough Bread Using Taguchi Grey Relational Analysis.

Bakery products made from naturally fermented sourdough show a diversified flavor and nutritional profile. Djulis (Chenopodium formosanum), known as red quinoa or Taiwan djulis, originally cultivated by Taiwanese indigenous people in mountain areas in eastern and southern Taiwan, has a high nutritional value and characteristic properties. In the present study, a new bakery product (djulis sourdough bread) was developed and a combination of the Taguchi method coupled with grey theory was utilized to optimize the baking parameters (product formulation). Five main factors, i.e., djulis sourdough (A), hulled djulis (B), oil type (C), a mixture of bread flour (wet gluten content of 29.0%) and a high-gluten flour (wet gluten content of 35.5%) (D), and honey (E), (each at four levels) were chosen for the Taguchi experiment design (L16(4)5). Dependent parameters were the data from texture profile analysis (brittleness, springiness, cohesiveness, gumminess, and chewiness), color analysis (L*, a*, and b*), and sensory evaluation (appearance, aroma, bitterness, sourness, chewiness, and overall acceptance) of the final product. Taguchi grey relational analysis successfully determined the optimal conditions based on combined parameters (5 factors), which highlighted the advantages of this innovative optimization technique. The result shows that the optimal formula for producing a djulis sourdough bread with the best texture, color, and sensory qualities was A3B1C1D2E2, i.e., 20% djulis sourdough, 0% addition of hulled djulis, 8% unsalted butter, 80% wheat flour + 20% high-gluten flour, and 10% honey, respectively. Such a novel application could be a reference for improving the quality of bakery products in the industry. Moreover, it seems that the new bakery product developed in this study has good potential to be commercially produced after further nutritional and economic analysis.

Chemical characterisation and histamine-forming bacteria in salted mullet roe products.

Sixteen salted mullet roe products sold in the retail markets in Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, water content, total volatile basic nitrogen (TVBN) and aerobic plate count (APC) in all samples ranged from 5.4 to 5.8, 5.1% to 7.2%, 15.4% to 27.3%, 32.0 to 69.6mg/100g and <1.0 to 7.1logCFU/g, respectively. None of these samples contained total coliform and Escherichia coli. The average content of each of the nine biogenic amines in all samples was less than 4mg/100g, and only one mullet roe sample had the histamine content (8.18mg/100g) greater than the 5.0mg/100g allowable limit suggested by the US Food and Drug Administration. Two histamine-producing bacterial strains capable of producing 10.7ppm and 9.6ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Staphylococcus carnosus by 16S rDNA sequencing with PCR amplification, and they were isolated from the sample with higher histamine content (8.18mg/100g).

Synthesis of terpinyl acetate by lipase-catalyzed esterification in supercritical carbon dioxide.

Terpinyl acetate was successfully synthesized from alpha-terpineol and acetic anhydride in supercritical carbon dioxide (SC-CO(2)) by enzymatic catalysis. Five different lipases (Candida rugosa type VII, Amano PS, Amano AP-6, Amano G and Lipozyme RM IM) as biocatalysts for the terpinyl acetate synthesis were compared. An esterification extent of 53.0% was obtained in continuous operation using acetic anhydride as acyl donor and C. rugosa lipase as enzyme at 10MPa and 50 degrees C for 1.5h. Temperature in the range of 35-50 degrees C demonstrated that the yield of terpinyl acetate increase with temperature increase in the current study. Operating at a alpha-terpineol/acetic anhydride molar ratio 3.0, the conversion of alcohol decreased, probably due to an inhibitory effect on enzyme by high concentration of acetic anhydride or by formation of acetic acid. However, the enzyme activity still remained more than 50% after 10.5h repeated esterification in a batch under optimized conditions.

High estrogen concentrations in receiving river discharge from a concentrated livestock feedlot.

Environmental estrogenic chemicals interrupt endocrine systems and generate reproductive abnormalities in wildlife, especially natural and synthetic estrogenic steroid hormones such as 17beta-estradiol (E2), estrone (E1), estriol (E3), 17alpha-ethynylestradiol (EE2), and diethylstilbestrol (DES). Concentrated animal feedlot operations (CAFOs) are of particular concern since large amounts of naturally excreted estrogens are discharged into aquatic environments. This study investigated E2, E1, E3, EE2, and DES with high performance liquid chromatography/tandem mass (HPLC-MS/MS) analyses along Wulo Creek in southern Taiwan, near a concentrated livestock feedlot containing 1,030,000 broiler chickens, 934,000 laying hens, 85,000 pigs, and 1500 cattle. Sampling was performed from December 2008 to May 2009, in which 54 samples were collected. Experimental results indicate that concentrations of EE2 were lower than the limit of detection (LOD), and concentrations of DES were only detected twice. Concentrations ranged from 7.4 to 1267 ng/L for E1, from not detected (ND) to 313.6 ng/L for E2, and from ND to 210 ng/L for E3. E1 had the highest average mass fraction (72.2 + or - 3.6%), which was significantly higher than E3 (16.2 + or - 1.7%) and E2 (11.5 + or - 2.6%). Additionally, the mean E2 equivalent quotient (EEQ) ranged from 17.3 to 137.9 ng-E2/L. Despite having a markedly lower concentration than E1, E2 more significantly contributed (52.4 + or - 6.0%) EEQ than E1 (19.7 + or - 3.5%). Moreover, the concentrations of E2, E1, and E3 upstream were significantly higher than concentrations downstream, suggesting a high attenuation effect and fast degradation in the study water. Most concentrations in winter season were higher than those of spring season due to the low dilution effect and low microbial activity in the winter season. Based on the results of this study, we recommend further treatment of the wastewater discharge from the feedlot.

Antihyperglycemic effect of polysaccharide from fermented broth of Pleurotus citrinopileatus.

Pleurotus citrinopileatus is an edible mushroom, which has recently become very popular, with a consequent increase in industrial production. Water-soluble polysaccharides (WSPS), extracted from edible mushrooms, have been found to have antitumor and immunoenhancing effects. In this study, we investigate the effects of WSPS extracted from submerged fermented medium of P. citrinopileatus on hyperglycemia and damaged pancreatic cells in rats with streptozotocin (STZ)-induced diabetes. The diabetic rats fed with water-soluble polysaccharide of P. citrinopileatus (SPPC) lost less body weight than those fed SPPC-free regular diet. Serum total cholesterol and triglyceride levels in the diabetic rats fed with SPPC at a dose of 0.4 g/kg bw daily was lower than in the groups fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The fasting blood glucose levels of diabetic rats fed with SPPC were 44% lower than the negative controls. The degree of damage to the islets of Langerhans of the rats fed with the highest dosage of SPPC was significantly lower than those fed with SPPC at doses of 0.04 and 0.12 g/kg bw. The results showed that STZ-induced diabetic rats fed with SPPC might help alleviate the elevation of the level of that in fasting blood glucose.