An improved method for estimating R-phycoerythrin and R-phycocyanin contents from crude aqueous extracts of Porphyra (Bangiales, Rhodophyta).
ABSTRACT: One frequently-cited method for determining phycoerythrin (PE) and phycocyanin (PC) contents from crude aqueous extracts of red seaweeds utilizes peaks and troughs of absorbance spectra. The trough absorbance values are used to establish a linear or logarithmic baseline attributable to background scatter of particulate cellular debris not removed by centrifugation. Pigment contents are calculated by subtracting baseline values from PE and PC absorbance peaks. The baseline correction is intended to make the method independent of centrifugation time and/or speed. However, when crude extracts of Porphyra were analyzed using this protocol, R-PE and R-PC estimates were significantly affected by centrifugation time, suggesting that the method was not reliable for the genus. The present study has shown that with sufficient centrifugation, background scatter in Porphyra extracts can be removed, the remaining spectrum representing the overlapping absorbance peaks of water-soluble pigments in the extract. Using fourth derivative analysis of Porphyra extract absorbance spectra, peaks corresponding to chlorophyll, R-PE, R-PC, and allophycocyanin (APC) were identified. Dilute solutions of purified R-PE, R-PC and chlorophyll were scanned separately to identify spectral overlaps and develop new equations for phycobilin quantification. The new equations were used to estimate R-PE and R-PC contents of Porphyra extracts and purified R-PE, R-PC and chlorophyll solutions were mixed according to concentrations corresponding to the sample estimates. Absorbances and fourth derivative spectra of the sample extract and purified pigment mixtures were compared and found to coincide. The newly derived equations are more accurate for determining R-PE and R-PC of Porphyra than previously published methods.
Project description:Crude extracts of fresh Dendrobium Sonia ‘Earsakul’ orchid flowers (DSE) were prepared using microwave assisted extraction (MAE; using household microwave oven) and hot water extraction (HWE; at constant 80?°C). The obtained DSEs were measured their absorbance at ?max of 543 and 583?nm and determined their total monomeric anthocyanin contents (TAC). Mathematical models of MAE of Dendrobium Sonia ‘Earsakul’ orchid flower were constructed using response surface methodology - Box-Behnken design. Studied parameters included flower to water ratio, microwave power, and extraction time, with absorbance at ?max as response. The data generated were 1) visible spectrum (400–700?nm) of DSE; 2) absorbance values at ?max and 3) TAC of DSEs obtained from various extraction conditions of MAE and HWE; 4) linear equations describing correlations between TAC and absorbance at ?max of DSEs; and 5) mathematical models of MAE of Dendrobium Sonia ‘Earsakul’ orchid.
Project description:Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) composition in skeletal muscle have been linked to insulin sensitivity. We evaluated the relationships between skeletal muscle PC:PE, physical exercise and insulin sensitivity. We performed lipidomics and measured PC and PE in m. vastus lateralis biopsies obtained from 13 normoglycemic normal weight men and 13 dysglycemic overweight men at rest, immediately after 45?min of cycling at 70% maximum oxygen uptake, and 2?h post-exercise, before as well as after 12 weeks of combined endurance- and strength-exercise intervention. Insulin sensitivity was monitored by euglycemic-hyperinsulinemic clamp. RNA-sequencing was performed on biopsies, and mitochondria and lipid droplets were quantified on electron microscopic images. Exercise intervention for 12?w enhanced insulin sensitivity by 33%, skeletal muscle levels of PC by 21%, PE by 42%, and reduced PC:PE by 16%. One bicycle session reduced PC:PE by 5%. PC:PE correlated negatively with insulin sensitivity (??=?-1.6, P?<?0.001), percent area of mitochondria (??=?-0.52, P?=?0.035), and lipid droplet area (??=?0.55, P?=?0.017) on EM pictures, and negatively with oxidative phosphorylation and mTOR based on RNA-sequencing. In conclusion, PC and PE contents of skeletal muscle respond to exercise, and PC:PE is inversely related to insulin sensitivity.
Project description:R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in <i>Porphyra yezoensis</i> with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A<sub>565</sub>/A<sub>280</sub>) of R-PE was 3.825, and the yield was 69.99% (<i>w/w</i>) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K<sub>2</sub>HPO<sub>4</sub> (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.
Project description:Algae contain a number of anti-inflammatory bioactive compounds such as omega-3 polyunsaturated fatty acids (n-3 PUFA) and chlorophyll a, hence as dietary ingredients, their extracts may be effective in chronic inflammation-linked metabolic diseases such as cardiovascular disease. In this study, anti-inflammatory potential of lipid extracts from three red seaweeds (Porphyra dioica, Palmaria palmata and Chondrus crispus) and one microalga (Pavlova lutheri) were assessed in lipopolysaccharide (LPS)-stimulated human THP-1 macrophages. Extracts contained 34%-42% total fatty acids as n-3 PUFA and 5%-7% crude extract as pigments, including chlorophyll a, ?-carotene and fucoxanthin. Pretreatment of the THP-1 cells with lipid extract from P. palmata inhibited production of the pro-inflammatory cytokines interleukin (IL)-6 (p < 0.05) and IL-8 (p < 0.05) while that of P. lutheri inhibited IL-6 (p < 0.01) production. Quantitative gene expression analysis of a panel of 92 genes linked to inflammatory signaling pathway revealed down-regulation of the expression of 14 pro-inflammatory genes (TLR1, TLR2, TLR4, TLR8, TRAF5, TRAF6, TNFSF18, IL6R, IL23, CCR1, CCR4, CCL17, STAT3, MAP3K1) by the lipid extracts. The lipid extracts effectively inhibited the LPS-induced pro-inflammatory signaling pathways mediated via toll-like receptors, chemokines and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) signaling molecules. These results suggest that lipid extracts from P. lutheri, P. palmata, P. dioica and C. crispus can inhibit LPS-induced inflammatory pathways in human macrophages. Therefore, algal lipid extracts should be further explored as anti-inflammatory ingredients for chronic inflammation-linked metabolic diseases.
Project description:Acetaminophen (APAP) overdose is one of the most common causes of acute liver failure in many countries. The aim of the study was to describe the profiling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in the plasma and liver of Acetaminophen -induced liver injured mice.A time course study was carried out using C57BL/6 mice after intraperitoneal administration of 300 mg/kg Acetaminophen 1 h, 3 h, 6 h, 12 h and 24 h. A high-throughput liquid chromatography mass spectrometry (LC-MS) lipidomic method was utilized to detect phosphatidylcholine and phosphatidylethanolamine species in the plasma and liver. The expressions of phosphatidylcholine and phosphatidylethanolamine metabolism related genes in liver were detected by quantitative Reverse transcription polymerase chain reaction (qRT-PCR) and Western-blot.Following Acetaminophen treatment, the content of many PC and PE species in plasma increased from 1 h time point, peaked at 3 h or 6 h, and tended to return to baseline at 24 h time point. The relative contents of almost all PC species in liver decreased from 1 h, appeared to be lowest at 6 h, and then return to normality at 24 h, which might be partly explained by the suppression of phospholipases mRNA expressions and the induction of choline kinase (Chka) expression. Inconsistent with PC profile, the relative contents of many PE species in liver increased upon Acetaminophen treatment, which might be caused by the down-regulation of phosphatidylethanolamine N-methyltransferase (Pemt).Acetaminophen overdose induced dramatic change of many PC and PE species in plasma and liver, which might be caused by damaging hepatocytes and interfering the phospholipid metabolism in Acetaminophen -injured liver.
Project description:Soybean sprouts are one of the most inexpensive and nutritious food items that can be easily grown year-round. Several studies have been conducted to increase their yield and nutritional values. This study was carried out to examine the effects of Pu-erh tea extracts on the production and nutrients content of soybean sprouts. Soybean seeds were soaked in 1%, 2%, or 3% (w/v) tea extracts, or tap water, before keeping for sprout cultivation; the sprout samples were named PE-1, PE-2, PE-3, and the control, respectively. The sprout yields were increased by up to 17% in PE-2 and PE-3 than in the control. The vitamin C, total free amino acid, total mineral, total isoflavone, total polyphenol, and flavonoid contents as well as the antioxidant potentials of the tea extract-treated sprouts were higher than those of the control. The results indicated that pre-soaking soybean seeds in 2% Pu-erh tea extracts could offer an easy, inexpensive, and efficient way to improve the yield and nutritional value of soybean sprouts.
Project description:1. A proteoglycan fraction (the proteoglycan subunit fraction) was prepared from extracts, with 0.15m-KCl (low-ionic-strength) and 0.5m-LaCl(3), 2.0m-CaCl(2) and 4.0m-guanidinium chloride (high-ionic-strength), of bovine nasal cartilage by equilibrium-density-gradient centrifugation, essentially as described by Hascall & Sajdera (1969). 2. The use of different centrifugation times showed that near-equilibrium conditions were reached by 48h for the fractions prepared from the high-ionic-strength extracts. The fraction isolated from the low-ionic-strength extract required a longer centrifugation time to reach equilibrium conditions. 3. The composition of the proteoglycan fractions from the various extracts was compared by analyses of their carbohydrate and amino acid contents. Difference indices were calculated from the amino acid analysis to compare the degree of compositional relationship between the protein components of the proteoglycans. 4. Small compositional differences were found between the proteoglycans isolated from the various high-ionic-strength extracts. The protein content of the fractions from the CaCl(2) extract and the guanidinium chloride extract showed the greatest difference in this respect, although their amino acid analysis was similar. 5. The proteoglycan fraction isolated from the low-ionic-strength extract shows marked differences in composition from the fractions isolated from the high-ionic-strength extracts. Its protein and glucosamine contents were lower whereas its hexuronic acid and galactosamine contents were higher than those of the latter. It also exhibits major differences in its amino acid composition. The glucosamine:galactosamine ratio of the fraction from the low-ionic-strength extract indicates that it may be an almost exclusively chondroitin sulphate-proteoglycan. Its analysis correlates closely with that of a low-molecular-weight proteoglycan isolated from pig laryngeal cartilage by Tsiganos & Muir (1969). 6. The proteoglycan fractions from both the low- and high-ionic-strength extracts migrate as a single band in zone electrophoresis carried out in a sucrose-density gradient at both pH3.0 and pH7.0, although each showed evidence of band widening during the electrophoresis. All the proteoglycan fractions migrated with the same electrophoretic mobility at pH3.0, irrespective of the differences in composition between them. 7. The differences between the proteoglycans from the low- and high-ionic-strength extracts are discussed and the view is advanced that they may be due to association between predominantly chondroitin sulphate-proteoglycans and a keratan sulphate-enriched proteoglycan species.
Project description:In the present study, methanol and aqueous extracts of Parkinsonia aculeata L. leaves were prepared and analyzed for phytochemical analysis and antioxidant potential in different in vitro assays. Antioxidant activity was studied using DPPH, CUPRAC, reducing power assay, deoxyribose degradation (site and nonsite specific), ferric reducing antioxidant potential (FRAP), ferric thiocyanate (FTC), thiobarbituric acid (TBA), and molybdate ion reduction, respectively. The total phenolic contents of the methanol and aqueous leaf extract were 39?mg?GAE/g and 38?mg?GAE/g, whereas flavonoid contents of these extracts were found to be 0.013?mg?RE/g and 0.006?mg?RE/g, respectively. From the two extracts, the methanol extract shows maximum inhibition (%) of 57.82%, 71.23%, 48.26%, 69.85%, and 52.78% in DPPH, nonsite- and site-specific, FTC, and TBA assays and absorbance of 0.669 and 0.241 in reducing power and CUPRAC assays at the highest concentration tested. UPLC analysis was done to determine the presence of various types of polyphenols present in plant extracts.
Project description:This publication reports high resolution mass spectral data for copper chlorophyll and copper chlorophyll degradation products extracted from bright green table olives. These data support analyte identifications made in "Quantitation of copper chlorophylls in green table olives by ultra-high-performance liquid chromatography with inductively coupled plasma isotope dilution mass spectrometry" in the Journal of Chromatography A (Petigara Harp et al., 2020 ). Table olive pigments, divided into lipophilic and hydrophilic fractions by liquid-liquid repartition, were separated by ultra-high-performance liquid chromatography and detected by visible wavelength absorbance and high resolution mass spectrometry, using an Orbitrap HF with positive electrospray ionization. Full-scan mass spectra were acquired to assign pigment chemical formulae. Fragment-rich higher-energy collisional dissociation tandem mass spectra were acquired to facilitate structural assignments. Extracted ion chromatograms, full-scan, and tandem mass spectra obtained from representative lipophilic and hydrophilic green table olive extracts are presented in Figures 1-6. Annotated mass spectra comparing experimental and calculated isotope distributions, .raw mass spectral data files, and experimental details linking .raw data files to annotated spectra are provided as Supplementary Material. Spectra extracted from these native data files can be added to mass spectral libraries for use in other studies. Access to native data files uniquely enables rigorous data examination (e.g., molecular ion isotopic distribution, effective mass resolution, presence of overlapping ion series) and use in ways that are not possible when spectra are otherwise reported in simple tables listing mono-isotopic peaks and mass errors. Mass spectra reported here can be used to design multiple-reaction monitoring methods to detect these bright green pigments in agricultural food commodities and finished products.
Project description:Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [(14)C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [(14)C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [(14)C]-LPC accumulated in HAEC as well. Its conversion to [(14)C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [(3)H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.