Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid chromatography-mass spectrometry using negative ion atmospheric pressure chemical ionization

Negative ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [negative ion LC-(APCI)MS] was used for the identification of astaxanthin esters in extracts of commercial shrimp (Pandalus borealis) and dried microalga (Haematococcus pluvialis) samples. A cleanup step using a normal phase solid phase extraction (SPE) cartridge was applied prior to analysis. Recovery experiments with astaxanthin oleate as model compound proved the applicability of this step (98.5 +/- 7.6%; n = 4). The assignment of astaxanthin esters in negative ion LC-(APCI)MS was based on the detection of the molecular ion (M*-) and the formation of characteristic fragment ions, resulting from the loss of one or two fatty acids. Quantification of individual astaxanthin esters was performed using an astaxanthin calibration curve, which was found to be linear over the required range (1-51 micromol/L; r2 = 0.9996). Detection limits, based on the intensity of M*-, a signal-to-noise ratio of 3:1, and an injection volume of 20 microL, were estimated to be 0.05 microg/mL (free astaxanthin), 0.28 microg/mL (astaxanthin-C16:0), and 0.78 microg/mL (astaxanthin-C16:0/C16:0), respectively. This LC-(APCI)MS method allows for the first time the characterization of native astaxanthin esters in P. borealis and H. pluvialis without using time-consuming isolation steps with subsequent gas chromatographic analyses of fatty acid methyl esters. The results suggest that the pattern of astaxanthin-bound polyunsaturated fatty acids of P. borealis does not reflect the respective fatty acid pattern found in triacylglycerides. Application of the presented LC-(APCI)MS technique in common astaxanthin ester analysis will forestall erroneous xanthophyll ester assignment in natural sources.     

Hydrolysis kinetics of astaxanthin esters and stability of astaxanthin of Haematococcus pluvialis during saponification

The reaction kinetics for the hydrolysis of astaxanthin esters and the degradation of astaxanthin during saponification of the pigment extract from the microalga Haematococcus pluvialis were investigated. Different concentrations of sodium hydroxide in methanol were used for the saponification under nitrogen in darkness at ambient temperature (22 degrees C) followed by the analysis of astaxanthins and other carotenoids using an HPLC method. The concentration of methanolic NaOH solution was important for promoting the hydrolysis of astaxanthin esters and minimizing the degradation of astaxanthin during saponification. With a higher concentration of methanolic NaOH solution, the reaction rate of hydrolysis was high, but the degradation of astaxanthin occurred significantly. The rate constants of the hydrolysis reaction (first order) of astaxanthin esters and the degradation reaction (zero-order) of astaxanthin were directly proportional to the concentration of sodium hydroxide in the saponified solution. Although the concentration of sodium hydroxide in the saponified solution was 0.018 M, complete hydrolysis of astaxanthin esters was achieved in 6 h for different concentrations (10-100 mg/L) of pigment extracts. Results also indicated that a higher temperature should be avoided to minimize the degradation of astaxanthin. In addition, during saponification, no loss of lutein, beta-carotene, and canthaxanthin was found.     

One-solvent extraction of astaxanthin from lactic acid fermented shrimp wastes

Free astaxanthin one-solvent extractions with ethanol, acetone, and liquid 1,1,1,2-tetrafluoroethane from raw and lactic acid fermented (ensilaged) shrimp residues were investigated. The total carotenoid recovery from ensilaged shrimp wastes was higher than that from non-ensilaged ones as assessed by HPLC analyses. Acetone gave the highest extraction yields of free astaxanthin with up to 115 microg/g of material. Moreover, liquid tetrafluoroethane is reported for the first time in a successful one-solvent extraction of carotenoids from shrimp.     

Determination of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) by liquid chromatography

The objectives of this study were to develop a high-performance liquid chromatography method for analysis of carotenoids in spear shrimp shells (Parapenaeopsis hardwickii) and to compare the extraction efficiency of carotenoids by supercritical carbon dioxide (SCD) and solvents. Results showed that the most appropriate HPLC method was accomplished by employing a Cosmosil 5C18-AR-II column and a mobile phase of methanol-dichloromethane-acetonitrile (90:5:5, v/v/v) (A) and water (100%) (B) with the following gradient elution: 92% A and 8% B in the beginning, decreased to 4% B in 9.5 min, 1% B in 26 min, 0% B in 35 min, maintained for 25 min, and returned to 92% A and 8% B in 61 min. All-trans-astaxanthin and its two cis isomers, as well as five astaxanthin monoesters and 11 diesters were resolved within 60 min with a flow rate at 2 mL/min and detection at 480 nm. Astaxanthin diesters were found to contain 12 fatty acids, of which palmitic acid and stearic acid constituted a large portion, whereas astaxanthin monoesters were found to contain 10 fatty acids with arachidonic acid dominating. Solvent extraction could generate a higher content of trans-astaxanthin and astaxanthin esters, while SCD extraction could produce greater levels of 9-cis-astaxanthin and 13-cis-astaxanthin.     

Wheat gluten phenolic acids: occurrence and fate upon mixing

Ferulic acid (FA, 4.9-17.7 microg/100 mg), sinapic acid (SA, 1.4-3.5 microg/100 mg), and traces of p-coumaric acid and vanillic acid were detected after saponification of six wheat glutens from industrial and pilot-scale origins. FA and SA occurred mostly as soluble-bound and insoluble-bound forms according to their extractability by acetone/methanol/water (7:7:6, v/v/v). The major part of FA (50-95%) was found in the unextractable fraction, whereas SA was mostly extractable (64-85%). The carbohydrate contents of the glutens were determined also after acid hydrolysis. The highest levels of glucose, arabinoxylan, and FA were obtained from the unextractable fractions of the pilot-scale extracted glutens, probably in relation with a lower efficiency of washing during extraction compared to industrial processes. On the other hand, SA compounds were in similar concentrations in all samples, suggesting their involvement in specific interactions during gluten protein agglomeration. Saponification of the soluble-bound phenolic acids released mainly glucose, whereas a beta-glucosidase treatment had no effect. FA and SA extractability, especially that of soluble-bound ones, decreased strongly in overmixed gluten/water doughs. These low molecular weight conjugates of phenolic acids could be involved in the dough breakdown phenomenon.     

Enzymatic extractability of soybean meal proteins and carbohydrates: heat and humidity effects

To study the incomplete enzymatic extractability of proteins and carbohydrates of thermally treated soybean meals, one unheated and three heat-treated soybean meals were produced. To obtain truly enzyme-resistant material, the meals were extracted by a repeated hydrolysis procedure using excessive concentrations of different combinations of commercial protease and carbohydrase preparations. The water extractability of protein from the different meals varied considerably (13-67%). For all soybean meals, enzymatic treatment extracted most of the original protein (89-94%). Carbohydrase preparations did not improve protein extraction. High-humidity heat treatment led to a more effective enzymatic extraction, which seemed to correlate with the extent of protein denaturation. Results with purified proteins indicated that the soybean meal matrix affects the enzymatic extraction of protein from the meals. Interactions between protein and other components (e.g., cellulose) may explain the incomplete enzymatic extractability of protein from the meals.     

Extractability of manganese and iron oxides in typical Japanese soils by 0.5 mol L−1 hydroxylamine hydrochloride (pH 1.5)

We investigated the extractability of manganese (Mn) and iron (Fe) oxides from typical Japanese soils (Entisols, Inceptisols, and Andisols) by 0.5?mol?L^?1 hydroxylamine hydrochloride (NH₂OH-HCl) extraction (pH 1.5; 16?h shaking at 25°C; soil:solution ratio 1:40), referred as to HH_mBCR, which is Step 2 (used for the reducible fraction) of the modified BCR (Community Bureau of Reference) sequential extraction procedure. The HH_mBCR procedure extracted almost all Mn oxides from the non-Andisol samples, but failed to extract a part of the Mn oxides from some Andisol samples. The procedure extracted most short-range ordered Fe oxides from non-Andisol samples, but it extracted only 7.5% and 13% of the short-range ordered Fe oxides from allophanic and non-allophanic Andisol samples, respectively. This remarkably low extractability of Fe oxides suggests that the HH_mBCR method is not suitable for extracting oxide-occluded heavy metals from Andisols. Since the extraction rate of short-range ordered Fe oxides from various soils with the extractant was negatively correlated with the amounts of oxalate- and pyrophosphate-extractable Al even when the variability of the extraction pH was reduced by increasing the soil:solution ratio from 1:40 to 1:500, the extractability of Fe oxides would be negatively affected by the presence of active Al, including allophane/imogolite, amorphous Al, and Al-humus complexes. Because these Al constituents are abundant in Andisols, they would be at least partially responsible for the lower extractability of Fe oxides by HH_mBCR from Andisols.     

Removal of Heavy Metals from Soil Components and Soils by Natural Chelating Agents. Part II. Soil Extraction by Sugar Acids

Aqueous solutions of the natural chelatingagents D-gluconic acid and D-glucaric acid (D[+]-saccharic acid) were tested for their ability to remove heavy metals (Cd, Cr, Cu,Ni, Pb, Zn) from a soil polluted by long-term application of sewage sludge. Batch equilibrium experiments were performed undervariation of fundamental process parameters, i.e. pH value, sugaracid concentration, batch solution volume, solid:liquid ratioand number of treatment cycles.The extractability of heavy metals was low under near-neutral andslightly basic pH conditions. It increased drastically between pH12.0 and 13.0. Pb and Cu were preferentially extracted metals.Compared with the extraction efficiency of pH adequate puresodium hydroxide solutions, the sugar acids enhanced thesolubilisation of Pb and Cr especially. The metal depletion fromsoil was the highest when applying 20 or 50 g L^-1 solutionsof the chelating agents. Under strongly basic conditions solid:liquid ratios of 1:10 or 1:20 were proofed to be advantageous.Except Ni, multi-step extraction improved the metal removalstrongly. This effect was the greatest for Cr extraction. Underoptimised conditions the following metal extraction degrees wereachieved with strongly alkaline D-gluconic acid solutions: Ni 43%%, Cr 60%%, Cd 63%%,Zn 70%%, Pb 80%%, and Cu 84%%.     

Feasibility of a cell separation-proteomic based method for soils with different edaphic properties and microbial biomass

The suitability of a soil proteome analysis based on previous cell extraction by gradient centrifugation was tested in semiarid soils with distinct edaphic properties and microbial biomass after enrichment with carbon and nitrogen. A sandy loam soil with low organic carbon content reached higher microbial biomass (estimated by PLFAs) after stimulation with nutrient sources (glucose and proline) than a naturally rich soil. However, the extractability of soil microbial cells was higher in a poor soil with high electrical conductivity probably due to the high saline content. The number of identified proteins in the poor soils reached 71 with proteins related to energy processes, transport and nucleic acid metabolism representing the highest percentage. High organic carbon content negatively influences cell extraction and protein separation and analysis. Soil texture and/or salinity might be related to the expression of proteins involved in the removal of reactive oxygen species (ROS) such catalase and superoxide dismutase (SOD) under active metabolism and microbial biomass development     

CO2和乙酸钠对雨生红球藻生长及其虾青素积累的影响

雨生红球藻(Haematococcus pluvialis)是强抗氧化剂虾青素的天然优质来源,碳源是影响雨生红球藻虾青素产量的重要因素之一。为探究CO₂和乙酸钠在雨生红球藻生长和虾青素积累中的作用,本研究通过测定生化指标和荧光定量PCR的方法,比较了这2种碳源对雨生红球藻干重、虾青素含量、生长相关酶及基因转录水平等的影响。结果表明,在绿色营养阶段,高CO₂组培养雨生红球藻干重为对照组的1.81倍(第8天),可溶性蛋白含量和核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)活力升高,磷酸烯醇式丙酮酸羧化酶(PEPC)和苹果酸脱氢酶(MDH)活力及其编码基因转录表达上调;乙酸钠组雨生红球藻干重为对照组的1.56倍(第8天),Rubisco活力及其大亚基转录表达受抑制,PEPC和MDH活力及其转录水平升高。在厚壁孢子阶段,高CO₂组雨生红球藻细胞状态良好,其干重和虾青素产量分别为对照组的1.96和2.40倍(第8天),3个虾青素合成相关酶编码基因的转录水平在第3天高于对照组,Rubisco、PEPC、MDH活力较高;乙酸钠组雨生红球藻干重和虾青素产量分别为对照组的1.54和1.85倍(第8天),高光胁迫1 d后藻细胞部分变红,同时3个虾青素合成相关酶的基因表达量快速升高、Rubisco活力降低,而PEPC和MDH活力升高。综上,补充CO₂或乙酸钠均可显著促进雨生红球藻生长和虾青素积累,而高CO₂浓度培养藻的状态较好,乙酸钠则可促进虾青素的快速积累。本研究结果为今后利用雨生红球藻高效生产虾青素提供了理论参考。     

Feasibility of a counter-current extraction procedure for the removal of heavy metals from contaminated soils

Effective remediation and sanitation technologies for soils contaminated with heavy metals are limited. We investigated the feasibility of a counter-current metal extraction procedure for the removal of selected heavy metals (Cd, Cu, Ph, and Zn) from two contaminated soils. The process involved a decarbonation (removal of carbonates), acid solubilisation, washing, and liming step. Results from batch equilibration experiments simulating the counter-current process showed more than 85% of the Cd present to be removed. Removal efficiencies for Cu and Pb were limited to approximately 15%, this mainly due to resorption of these elements during the decarbonation step. As most Zn was found to be present in a more difficult acid-extractable solid phase, its extractability accounted for only 25%. While reaction (pH) conditions of both decarbonation and solubilisation determined removal efficiencies, washing the extracted soil with deionized water only slightly increased the amount of metals removed. Metal distribution among solid phases — exchangeable, carbonate, reducible, organically bound, and residual — was affected by the different treatments. The amount of metals contained in the exchangeable and residual fractions determined their extractability. Except for Cu, the reducible and organically bound fractions were less important. After solubilisation 13 to 70% of the metals were present in an exchangeable solid phase. This implicates that washing the solubilized soil with a salt may increase the extractability of metals, especially for Zn and Pb. Based on our results the process is critically evaluated and possibilities for optimization formulated.     

Extractability of 2,4-D,Dicamba and MCPP from Soil

The adsorption of herbicides on soil colloids is a major factor determining their mobility, persistence, and activity in soils. Solvent extraction could be a viable option for removing sorbed contaminants in soils. This study evaluated the extractability of three herbicides: 2,4 dichlorophenoxy-acetic acid (2,4-D), 4-chloro-2-methylphenoxypropanoic acid (mecoprop acid or MCPP), and 3,6-dichloro-2-methoxybenzoic acid (dicamba). Three solvents (water, methanol, and iso-propanol) and three methods of extraction (column, batch, and soxhlet) were compared for their efficiencies in removing the herbicides from three soils (loamy sand, silt loam, and silty clay). Both linear and non-linear Freundlich isotherms were used to predict sorption intensity of herbicides on soils subjected to various extraction methods and conditions. High K_dand K_fr, and low N values were obtained for all herbicides in silty clay soil by batch extraction. Methanol was the best solvent removing approximately 97% of all added herbicides from the loamy sand either by column or soxhlet extraction method. Isopropanol ranked second by removing over 90% of all herbicides by soxhelet extraction from all three soils. However, water was ineffective in removing herbicides from any of the soils using any of the three extracting procedures used in this study. In general, the extent of herbicide removal depended on soil type, herbicide concentration, extraction procedure, solvent type and amount, and extraction time.     

Carotenogenesis up-regulation in Scenedesmus sp. using a targeted metabolomics approach by liquid chromatography-high-resolution mass spectrometry

Carotenoids have potent antioxidant activity as well as therapeutic value, and their formation has been seen to be induced in algae by stress, including high-salt culture conditions. A differential profiling of carotenoids was conducted using a targeted metabolomics approach with accurate mass data generated by liquid chromatography-electrospray-time-of-flight (LC-ESI-TOF) mass spectrometry followed by postacquisition filtering based on isotope patterns and mass defects to detect carotenoids up-regulated in Scenedesmus sp. exposed to high-salt conditions. Algal cultures treated with high concentrations of sodium acetate or sodium chloride were found to cause an increase in various carotenoids. On the basis of differential analysis, astaxanthin and canthaxanthin increased upon salt treatment. Astaxanthin, in its free form and as fatty acid esters, was seen to increase in Scenedesmus sp. using accurate mass MS. A few other carotenoid compounds increased upon salt treatment, including echinenone and adonirubin, involved in the pathway of astaxanthin biosynthesis from β-carotene, as well as isomers of astaxanthin and canthaxanthin. A time course study of acetate treatment was done to observe the time-dependent up-regulation of carotenogenesis.     

Effect of Organic Matter on the Release Behavior and Extractability of Copper and Cadmium in Soil

Land disposal of organic waste materials may alter the heavy-metal status of the soil by affecting metal solubility or dissociation kinetics. The aim of this investigation was to study the influences of poultry manure and pistachio compost (at the rate of 300 g kg^?1) on release behavior and extractability of copper (Cu) and cadmium (Cd) in two soils of Iran. For release studies, samples after incubation were extracted with ethylenediaminetetraacetic acid (EDTA) at pH 7.0 with shaker for periods of 5 to 2880 min. Results showed that during the reaction periods, Cu and Cd release rates were rapid at first and then became slower until equilibrium was achieved. In the two soils, extractability of Cu increased with poultry manure treatment and decreased with pistachio compost treatment as compared to the control soil, and extractability of Cd decreased with both pistachio compost and poultry manure treatments as compared to the control soil.     

超声波辅助碱分离毛竹半纤维素

该文以高效分离毛竹半纤维素为出发点,采用超声辅助碱分离法分离毛竹半纤维素。在固定固液比1∶20g/mL、总浸提时间120min的条件下,分析了KOH溶液质量分数,浸提温度及超声时间对毛竹半纤维素得率的影响。在单因素试验的基础上,采用Box-Behnken试验设计和通过响应面分析对分离工艺进行了优化。结果表明,超声辅助碱分离毛竹半纤维素的最佳分离工艺参数为KOH溶液质量分数3.1%、浸提温度54℃、超声时间56min,此时半纤维素实际总得率为19.28%。浸提温度和KOH溶液质量分数对半纤维素总得率影响显著,超声波作用时间对总得率影响不显著。在相同条件下超声辅助碱分离时半纤维素提取率比无超声辅助时提高4.06%。结果可为毛竹半纤维素分离的工业化提供参考。     

Protein Extraction from Triticale Distillers Grains

Triticale is being actively explored as a feedstock for bioethanol production in Western Canada. Triticale distillers grains, an important coproduct of the bioethanol industry, are used mainly as animal feed. This study aims to develop methods of protein extraction from triticale distillers wet grains and distillers dried grains with solubles. Osborne fractionation showed low protein extractability because excessive protein denaturation occurred during sample preparation. Five methods were used to extract proteins: pH shifting, 60% ethanol, alkaline‐ethanol solution, glacial acetic acid, and enzyme‐aided extraction. Extracts obtained with the alkaline‐ethanol and glacial acetic acid methods showed comparatively higher protein contents (≈61–65%) when compared with the other extraction methods (≈35–57%). Enzyme‐aided extraction with Protex 6L yielded 75–82% protein at a content of 43–57%, depending on the types of raw materials. Establishing methods of protein extraction from triticale distillers grains would facilitate further studies on new uses of triticale proteins.     

利用农业废弃物碳源的红法夫酵母生产虾青素研究进展

利用红法夫酵母产虾青素工艺具有绿色、安全、低成本等优点,但培养红法夫酵母的培养基(尤其是碳源)成本是制约该工艺实现大规模生产的主要因素之一。传统的利用纯糖如葡萄糖、蔗糖、木糖等作为红法夫酵母生产虾青素的碳源,效果虽好,但因其价格较高,仅适用于实验室研究和小型发酵生产,不适于大规模工业化生产。如何寻找来源广、价廉且发酵效果好的碳源,降低虾青素的生产成本是发展虾青素产业亟待解决的课题。利用廉价且来源广泛的原料如水果类原料、糖质原料、甘油及废弃木质纤维素类原料等作为碳源能有效降低成本。但这些原料都存在一些问题,比如水果类原料存在季节性及地域性限制,糖蜜类原料供应稳定性方面存在问题,甘油类原料杂质多且存在食品安全隐患,木质纤维素类原料来源广泛、价格低廉,不存在占用耕地等问题,是最有希望用于规模化低成本发酵生产虾青素的原料,但其利用存在一些技术瓶颈。该文介绍了目前利用红法夫酵母生产虾青素的基本情况,并系统地对国内外不同碳源条件下利用红法夫酵母生产虾青素的研究状况进行了综述、比较及分析。最后,对利用红法夫酵母生产虾青素在碳源方面的未来发展趋势及重点研究方向进行了展望。     

Use of 1,10‐phenanthroline in estimating metabolically active iron in plants

Abstract

If calcifuges are forced to grow on a calcareous soil, they usually develop chlorosis. However, total leaf iron (Fe) does not often correlate well with Fe deficiency symptoms. The extraction of ‘active’ Fe by 1 M HCl or Fe chelators, e.g., 1,10‐phenanthroline, may reflect the relation between chlorosis and Fe‐concentration in the leaves better than total Fe does. Extraction of ‘active’ Fe from leaves of wild plants by 1,10‐phenanthroline, citric acid and HC1 was compared. The 1,10‐phenanthroline was chosen for further methodological studies. All samples were extracted at indoor light conditions and analyzed by AAS because dark incubation did not influence the oxidation state of Fe and non‐specific light absorbance seemed to be high in colorimetric analysis. Washing of leaf material with H₂O seemed to clean the leaf surfaces equally well as with 0.1 M HCl. Only fresh leaf material was extracted, as pretreatment (freezing or drying) changed the extractability of Fe. An extraction time of 16 h was adequate for the herbaceous plants tested but not for Carex pilulifera, where extracted Fe increased linearly with time. The age of the extractant solution may play a role because 1,10‐phenanthroline had lost part of its chelation capacity after 6 weeks. The ratio of leaf weight:extractant volume did not influence the amount of Fe extracted, provided the same amount of chelator was supplied. The 1,10‐phenanthroline did not interfere with the Fe determination by AAS, and HCl pH 3 as used for the preparation of the extractants had only a marginal influence on Fe extractability compared to 1,10‐phenanthroline at pH 3. To get comparable results the extraction method should be standardized as much as possible. Samples can be stored in the refrigerator for several hours before adding the extractant and the extracts can be stored for a few days or frozen and measured on the same day, with the same instrument setting.     

阴离子对加入到土壤中的铅吸附与提取的影响

The effect of accompanying anions on the adsorbability and extractability of Pb added in soils was studied by using bach equilibria and incubation-extraction mothods.Pb concentration ranged between 0.48-2.41 mM in the adsorption experiment,and Pb added to soil from 2.14 to 19.31mM Pb/kg with an incubation time of 2-16 weeks in the incubation-extraction test in which 1M NH4OAc(pH 7) was taken as an extraction agent,and the Pb compounds used are PbCl2,Pb(NO3)2 and Pb(OAc)2.The values of pH50 for Pb retention and the parameters,b and k values,of Langmuir equation were used to distinguish the difference of adsorbability and extractability of Pb affected by anions,respectively.Judging from the results of either pH50 value or b value,the influences of Cl^- and NO3^- on the adsorbability and extractability of Pb in soils were similar but quite different from those of OAc^-.The relative loading capacities of soils to Pb calculated according to the extraction rate showed that the values of Pb(OAc)2 were higher than those of PbCl2 and Pb(NO3)2.     

Antioxidant activities of astaxanthin and related carotenoids

The antioxidant activities of astaxanthin and related carotenoids have been measured by employing a newly developed fluorometric assay. This assay is based on 4,4-difluoro-3,5-bis(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene (BODIPY 665/676) as an indicator; 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) as a peroxyl radical generator; and 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator in an organic and liposomal media. By employing this assay, three categories of carotenoids were examined: namely, the hydrocarbon carotenoids lycopene, alpha-carotene, and beta-carotene; the hydroxy carotenoid lutein; and the alpha-hydroxy-ketocarotenoid astaxanthin. The relative peroxyl radical scavenging activities of Trolox, astaxanthin, alpha-tocopherol, lycopene, beta-carotene, lutein, and alpha-carotene in octane/butyronitrile (9:1, v/v) were determined to be 1.0, 1.0, 1.3, 0.5, 0.4, 0.3, and 0.2, respectively. In dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tri-HCl buffer (pH 7.4 at 40 degrees C), the relative reactivities of astaxanthin, beta-carotene, alpha-tocopherol, and lutein were found to be 1.00, 0.9, 0.6, and 0.6, respectively. When BODIPY 665/676 was replaced by 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C(11)) as an indicator, astaxanthin showed the highest antioxidant activity toward peroxyl radicals. The relative reactivities of Trolox, astaxanthin, alpha-tocopherol, alpha-carotene, lutein, beta-carotene, and lycopene were determined to be 1.0, 1.3, 0.9, 0.5, 0.4, 0.2, and 0.4, respectively.