Efficient Isolation of Mycosporine-Like Amino Acids from Marine Red Algae by Fast Centrifugal Partition Chromatography

Marine rhodophyta are known to synthesize specific secondary metabolites, mycosporine-like amino acids (MAAs), to protect themselves from harmful UV-radiation. Shinorine and porphyra-334 are among the most abundant representatives of this compound class. In the present work, a novel approach for their isolation is described. As a first step, a fast centrifugal partition chromatography method, with an aqueous two-phase system comprising water, ethanol, ammonium sulfate and methanol in ascending mode, was developed to isolate the two MAAs from crude aqueous-methanolic extracts of three algal species within 90 min. The compounds could be isolated when just one of them was present in a sample or also both at the same time. By employing solid phase extraction as a second purification step, the individual MAAs were obtained in high purity and good quantity within a much shorter time frame than the established purification protocols, e.g., semi-preparative HPLC. For example, from 4 g Porphyra sp. (Nori) crude extract, 15.7 mg shinorine and 36.2 mg porphyra-334 were isolated. Both were highly pure, as confirmed by TLC, HPLC-MS and NMR analyses.     

Mycosporine-Like Amino Acids from Marine Resource

In the last 10 years, a great number of publications (both regular papers and reviews) have been published on the interesting molecules—mycosporine-like amino acids (MAAs). Despite significant advances in the research of MAAs, current overviews in the recent publications involving MAA research still need reporting. The aim of this Special Issue is to join, as an interdisciplinary approach, the photochemical and photobiological aspects, with emphasis on new natural resources to obtain both algae and zooplankton MAAs, advances in methodology of extraction and chemical identification of new MAAs. Finally, this Special Issue reviews the bioactivities of MAAs including UVR screen, antioxidant, immunostimulant, growth factor, DNA protection, inhibition of collagenase, elastase and hyaluronidase, and anti-photoaging, among others, and their potential use as nutracosmeceutic molecules (i.e., oral and topic photoprotector).     

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.     

Analysis of the Mycosporine-Like Amino Acid (MAA) Pattern of the Salt Marsh Red Alga Bostrychia scorpioides

This study presents the validation of a high-performance liquid chromatography diode array detector (HPLC-DAD) method for the determination of different mycosporine-like amino acids (MAAs) in the red alga Bostrychia scorpioides. The investigated MAAs, named bostrychines, have only been found in this specific species so far. The developed HPLC-DAD method was successfully applied for the quantification of the major MAAs in Bostrychia scorpioides extracts, collected from four different countries in Europe showing only minor differences between the investigated samples. In the past, several Bostrychia spp. have been reported to include cryptic species, and in some cases such as B. calliptera, B. simpliciuscula, and B. moritziana, the polyphyly was supported by differences in their MAA composition. The uniformity in the MAA composition of the investigated B. scorpioides samples is in agreement with the reported monophyly of this Bostrychia sp.     

Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan

We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8–8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.     

Analysis of Mycosporine-Like Amino Acids in Selected Algae and Cyanobacteria by Hydrophilic Interaction Liquid Chromatography and a Novel MAA from the Red Alga Catenella repens

Mycosporine-like amino acids (MAAs), a group of small secondary metabolites found in algae, cyanobacteria, lichens and fungi, have become ecologically and pharmacologically relevant because of their pronounced UV-absorbing and photo-protective potential. Their analytical characterization is generally achieved by reversed phase HPLC and the compounds are often quantified based on molar extinction coefficients. As an alternative approach, in our study a fully validated hydrophilic interaction liquid chromatography (HILIC) method is presented. It enables the precise quantification of several analytes with adequate retention times in a single run, and can be coupled directly to MS. Excellent linear correlation coefficients (R² > 0.9991) were obtained, with limit of detection (LOD) values ranging from 0.16 to 0.43 µg/mL. Furthermore, the assay was found to be accurate (recovery rates from 89.8% to 104.1%) and precise (intra-day precision: 5.6%, inter-day precision ≤6.6%). Several algae were assayed for their content of known MAAs like porphyra-334, shinorine, and palythine. Liquid chromatography-mass spectrometry (LC-MS) data indicated a novel compound in some of them, which could be isolated from the marine species Catenella repens and structurally elucidated by nuclear magnetic resonance spectroscopy (NMR) as (E)-3-hydroxy-2-((5-hydroxy-5-(hydroxymethyl)-2-methoxy-3-((2-sulfoethyl)amino)cyclohex-2-en-1-ylidene)amino) propanoic acid, a novel MAA called catenelline.     

Characterization of Novel Selected Microalgae for Antioxidant Activity and Polyphenols,Amino Acids,and Carbohydrates

The biochemical composition of three novel selected microalgae strains (Chlorophyta) was evaluated to confirm their potential possibilities as new sustainably produced biomass with nutritional, functional, and/or biomedical properties. Extracts from cultured Pseudopediastrum boryanum, Chloromonas cf. reticulata, and Chloroidium saccharophilum exhibited higher radical scavenging activity of DPPH (1,1-diphenyl-2-picrylhydrazyl) when compared to butylated hydroxytoluene (BHT), but lower than butylated hydroxyanisole (BHA). Total phenolic compounds and amino acids were determined by newly developed RP-HPLC methods. Total phenolic contents, as µg g⁻¹ of dry biomass, reached 27.1 for C. cf. reticulata, 26.4 for P. boryanum, and 55.8 for C. saccharophilum. Percentages of total analysed amino acids were 24.3, 32.1, and 18.5% of dry biomass, respectively, presenting high values for essential amino acids reaching 54.1, 72.6, and 61.2%, respectively. Glutamic acid was the most abundant free amino acid in all microalgae samples, followed by proline and lysine in C. saccharophilum and P. boryanum, and methionine and lysine in C. reticulata. Soluble carbohydrates in aqueous extracts ranged from 39.6 for C. saccharophilum to 49.3% for C. reticulata, increasing values to 45.1 for C. saccharophilum and 52.7% for P. boryanum in acid hydrolysates of dried biomass. Results confirmed the potential possibilities of these microalgae strains.     

邻苯二甲醛柱前衍生反相高效液相色谱法测定茶叶中17种游离氨基酸

采用邻苯二甲醛作为柱前衍生化试剂,结合反相高效液相色谱,优化了色谱条件,实现对茶汤中17种一级氨基酸的精确定性和定量分析。17种氨基酸的保留时间和峰面积的RSD分别在0.02%~0.70%与0.11%~1.15%之间,经方法学考察,该方法具有良好的稳定性和重现性。     

GC-MS及GC测定茶叶中主要游离氨基酸的方法研究

研究了N-（特丁基二甲基硅烷）-N-甲基三氟乙酰胺（≥95%;MTBSTFA）含叔丁基二甲基氯硅烷（1%;TBDMSCl）衍生、气相色谱（GC）和气相质谱联用仪（GC-MS）测定茶叶中游离氨基酸的方法,比较了衍生温度、时间、酸度及辅助试剂等对衍生效率的影响。以正缬氨酸为内标,乙腈为辅助衍生试剂,衍生温度110℃,衍生时间30 min,可以定量检测茶叶中主要的游离氨基酸。茶氨酸产生3个衍生峰,经质谱鉴定第1个峰（按出峰时间）为环状衍生峰,第2个峰为一个N端没有衍生,第3个峰为完全衍生峰。各氨基酸在10~1 000 nmol范围内线性良好（相关系数均大于0.99）,对茶叶样品测定结果与氨基酸自动分析仪基本一致,添加回收试验表明,茶氨酸、谷氨酸、谷氨酰胺、丝氨酸、天冬氨酸等的回收率为85%~108%。结果表明,试验建立的GC-MS及GC-FID方法操作简便、准确,重现性好,适用于茶叶中主要游离氨基酸的分离检测,同时GC-MS方法还可用于¹⁵N标记的氨基酸的检测,为吸收代谢实验提供研究基础。     

Subcritical Water Hydrolysis of Comb Pen Shell (Atrina pectinata) Edible Parts to Produce High-Value Amino Acid Products

Artina pectinata (Comb pen shell, CPS) is a high-protein source that contains a variety of essential amino acids. Subcritical water hydrolysis (SWH) was used to recover amino acids from the posterior adductor muscle (PAM), anterior adductor muscle (ADM), and mantle. The temperatures ranged from 120 °C to 200 °C, and the pressure and time of hydrolysis were 3 MPa and 30 min, respectively. Further characterization of the hydrolysates was performed to ascertain amino acid profiles and biofunctional properties. The hydrolysates contained more free amino acids than the untreated samples. Antioxidant activity of treated samples increased as SW temperatures increased. At 200 °C, those inhibiting ACE had a maximum antihypertensive activity of 200 °C in 1% PAM, ADM, and mantle with 85.85 ± 0.67, 84.55 ± 0.18, and 82.15 ± 0.85%, respectively, compared to 97.57 ± 0.67% in 1% standard captopril. Perhaps the most significant finding was the predominance of taurine in the three parts following SW treatment at 120 °C. The hydrolysates may be of considerable interest for use in food or energy drinks. SWH demonstrates efficacy in recovering amino acids, particularly taurine, from edible parts of A. pectinata.     

野生大豆苯丙氨酸解氨酶(PAL)在不同诱导条件下变化规律

以野生大豆(龙野01-177)为材料,根据PAL酶活性的影响因素,选取48 h为一个周期,每3h进行取样,研究低温(-4℃)诱导、机械损伤诱导、紫外线照射诱导、激素BAP诱导和正常对照组条件下的酶活性、蛋白含量、酶比活性的变化.结果表明:4种诱导条件下野生大豆PAL的酶活性和蛋白质含量变化趋势均相同且表现出不同的变化规...     

花蓟马对11种挥发性化合物的室内趋性

为明确不同信息化合物对花蓟马的行为趋性,采用“Y”型嗅觉仪测定了花蓟马雌成虫对11种挥发性化合物及其不同浓度(V/V)的行为反应。结果表明：花蓟马对10-6烟酸乙酯、水杨醛、芳樟醇和10-4苯甲醛、月桂烯以及10-2月桂烯都具有极显著的趋向反应;对10-6橙花醇和10-4邻茴香醛也具有显著的趋向反应;相反,10-6 β-香茅醇则对花蓟马雌成虫具有显著的驱避反应。不同浓度同一化合物对花蓟马雌成虫的吸引作用也存在显著差异。试验结果可为进一步开发利用信息化合物有效监测和防治花蓟马提供科学依据。     

大豆对大豆胞囊线虫侵染的应答机制研究

大豆胞囊线虫病(Heterodera glycines,soybean cyst nematode,SCN)是大豆生产上的重要病害,其特点为危害重、分布广、难防治,每年对大豆生产造成极大的损失。种植大豆抗性新品种是防治SCN目前最为有效的措施,研究大豆对SCN侵染的应答机制,是培育大豆持久抗病品种的前提,对加快抗线虫品种选育及SCN的防控具有重要的意义。本文综述了大豆对SCN侵染的组织细胞学应答机制;介绍了大豆在SCN侵染后酶系变化及酚类代谢的生理生化应答机制;从分子水平阐明了SCN侵染后大豆的基因转录变化,差异蛋白及DNA甲基化的应答机制,以期为大豆胞囊线虫病害的进一步研究与防治提供参考。     

温度对不同叶龄长雄野生稻及其后代化感作用的影响

掌握温度对长雄野生稻(Oryza longistaminata)及其后代化感作用的影响,对开发利用野生种质资源的化感抗草基因具有重要意义。采用直接树脂吸收法收集不同叶龄长雄野生稻及其后代在各温度条件下的根系分泌物,并以稗草作为受体,测定其化感作用。结果表明:1)温度、叶龄和温度×叶龄对水稻化感作用的影响与其自身化感潜力正相关,且对强化感和中化感潜力水稻(OL、F1、RL159和RL169)的化感作用影响极显著,而对弱化感水稻(RD23和RL219)的化感作用影响不显著。2)供试条件下长雄野生稻及其后代根系分泌物对稗草的化感作用均为抑制作用,其中,低温不利于水稻化感潜力的发挥。15℃时,强和中化感潜力水稻在不同叶龄对稗草的苗高、根长和生物量的抑制作用最弱,化感综合效应指数也显著大于同等叶龄的高温处理。3)高温有利于水稻化感潜力的发挥,但并非总是随温度的升高而增强。在6叶期和8叶期,水稻材料OL、F1、RL159和RL169随温度的升高化感作用逐渐增强,但在2叶期和4叶期则表现为先增强后减弱,在25℃时对稗草化感作用最强。4)在15℃时,强和中等化感潜力水稻在2叶期化感作用最弱,而在其他温度条件下,其化感作用随叶龄的增加表现为先减弱后增强,在2叶期化感作用最强。研究结果显示,温度对不同叶龄的水稻化感作用具有调控作用,幼苗阶段高温有利于强和中化感潜力水稻化感作用的发挥。     

冀东野生大豆对大豆花叶病毒的抗性鉴定及抗病反应

对冀东地区收集的162份野生大豆(Glycine soja)材料进行大豆花叶病毒(SMV)抗性鉴定并对筛选到的抗、感材料植株体内的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)活性及病程相关基因GmPR-1和GmPR-10的表达进行测定。结果表明:抗病野生大豆材料7份(4.3%),中抗材料6份(3.7%),中感材料62份(38.3%),感病材料87份(53.7%);抗病材料POD和CAT活性比对照显著增加,而感病材料显著降低,抗病材料的GmPR-1和GmPR-10基因表达量比对照显著增加,感病材料与对照无显著差异,初步表明POD、CAT及病程相关基因GmPR-1和GmPR-10可能与大豆抗病性有关。     

Response of Leaf Photosynthesis to Vapor Pressure Difference in Rice (Oryza sativa L) Varieties in Relation to Stomatal and Leaf Internal Conductance

Abstract

In the afternoon when air humidity decreases, leaf photosynthetic rate (P_n) often declines in rice grown under irrigated conditions. To clarify the genotypic difference of P_n in response to humidity, we measured P_n and stomatal conductance (g_s) for nine rice varieties with diverse genetic backgrounds, at various vapor pressure differences (VPD) and developmental stages. P_n and g_s of all the varieties decreased with VPD increase from 1.0 to 2.3 kPa of VPD. The variety with high g_s at low VPD exhibited a greater decline of g_s with VPD increase than the variety with low g_s, but cv. Takanari showed the highest g_s under altered VPD conditions. Significant logarithmic relations were found between the decreased P_n and g_s at the respective developmental stages, suggesting that g_s is the dominant factor determining P_n and its response to VPD change. To explicate the effect of decreased g_s on P_n, we analyzed the relations by using the model that accurately estimated the genotypic difference in P_n at a low VPD with g_s and leaf nitrogen content per unit leaf area in the previous study. The model assuming that leaf internal conductance (g_w) remains unchanged well explained the decreased P_n at high VPDs by g_s change alone. The analysis also suggested the constancy of g_w and carboxylation capacity at high VPD. It is concluded that the genotypic difference in the decrease of P_n at a high VPD is brought mainly by that in decreased g_s, and the varieties with a high g_s always exhibit a high P_n owing to their relatively high g_s at either high or low VPD environments.     

小麦籽粒多酚氧化酶（PPO）检测方法的优化及其在育种中的应用

降低小麦中多酚氧化酶(PPO)活性,减缓面粉制品的褐化,是重要的育种目标之一。为了更好地服务于低PPO育种,本研究对检测PPO活性的原苯酚染色法进行了优化,更好地发挥了其鉴别力强、结果稳定、对种子活力伤害小等优点,便于育种者使用。苯酚染色和分子标记结果对比发现,染色结果可以很好地反映亲本(或高代)材料中PPO的基因型,特别在低PPO材料中吻合更好。对大量亲本和世代材料的籽粒染色发现,PPO不仅存在于种皮中,其活性还是由种皮基因型决定的,后代PPO性状表现出母性遗传和加性效应的特点,控制高PPO特性的两个主效基因之间具有明显的代偿作用。PPO性状遗传相对简单,纯合较快,F2以后籽粒的染色程度以单株为单位发生分离。尽管染色是针对种皮基因型的,但PPO基因的这些遗传特点和小麦的自交特性,使染色结果同样可以预测后代单株的分离前途。这一优化的籽粒染色法在低PPO育种中的有效性是可以肯定的。     

Analysis of QTLs for Flag Leaf Shape and Its Response to Elevated CO2 in Rice （Oryza sativa）

To understand the responses of flag leaf shape in rice to elevated CO2 environment and their genetic characteristics,quantitative trait loci(QTLs)for flag leaf shape in rice were mapped onto the molecular marker linkage map of chromosome segment substitution lines(CSSLs)derived from a cross between a japonica variety Asominori and an indica variety IR24 under free air carbon dioxide enrichment(FACE,200μmol/mol above current levels)and current CO2 concentration(Ambient,about 370μmol/mol).Three flag-leaf traits,flag-leaf length(LL),width(LW)and the ratio of LL to LW (RLW),were estimated for each CSSL and their parental varieties.The differences in LL,LW and RLW between parents and in LL and LW within IR24 between FACE and Ambient were significant at 1%level.The continuous distributions and transgressive segregations of LL,LW and RLW were also observed in CSSL population,showing that the three traits were quantitatively inherited under both FACE and Ambient.A total of 16 QTLs for the three traits were detected on chromosomes 1,2,3,4,6,8 and 11 with LOD(Log10-likelihood ratio)scores ranging from 3.0 to 6.7.Among them,four QTLs (qLL-6*,qLL-8*,qLW-4*,and qRLW-6*)were commonly detected under both FACE and Ambient.Therefore,based on the different responses to elevated CO2 in comparison with current CO2 level,it can be suggested that the expressions of several QTLs associated with flag-leaf shape in rice could be induced by the high CO2 level.     

光敏核不育水稻的光温反应研究：Ⅱ．人工控制条件下粳型光敏不育系的育性鉴定

 在人工控制条件下研究了光照时间和温度对农垦58S及以此为基因源转育的其他粳型光敏不育系育性的影响。结果表明，供试粳型光敏不育系的育性受光照时间和温度的双重影响， 在15.0 h光长与29.6℃平均温度(最高与最低温度分别为33.0℃与28.0℃)的长光照高温条件下，自交结实率为0或接近于0；在12.0 h光长与23.6℃平均温度(27.0／22.0℃)的短光照低温条件下， 自交结实率较高；在长光低温(15.0 h／23.6℃)和短光高温(12.0 h／29.6℃)下，育性出现不同程度的转变。不同材料的育性对光温反应存在着差异，提示遗传背景对育性表达可能有重要作用。本研究结果有助于对自然条件下光敏不育系夏季制种纯度不高而秋季繁种产量又较低的现象在理论上给予合理的解释。     

光敏核不育水稻的光温反应研究：I．光敏核不育水稻在杭州(30°05 ′N) 自然条件下的育性表现

 通过大田分期播种，对现有主要光敏核不育水稻在杭州自然条件下的育性表达及其转换作了观察鉴定。结果表明，在杭州自然条件下，光敏核不育水稻的光温反应因遗传背景不同而异，育性转换的时间也不一致。温度在光敏核不育水稻育性表达与转换中起重要作用，对于籼型材料甚至占主导作用。