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.     

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.     

Complementary UV-Absorption of Mycosporine-like Amino Acids and Scytonemin is Responsible for the UV-Insensitivity of Photosynthesis in Nostoc flagelliforme

Mycosporine-like amino acids (MAAs) and scytonemin are UV-screening compounds that have presumably appeared early in the history of life and are widespread in cyanobacteria. Natural colonies of the UV-insensitive Nostoc flagelliforme were found to be especially rich in MAAs (32.1 mg g DW⁻¹), concentrated in the glycan sheath together with scytonemin. MAAs are present in the form of oligosaccharide-linked molecules. Photosystem II activity, measured using PAM fluorescence and oxygen evolution, was used as a most sensitive physiological parameter to analyse the effectiveness of UV-protection. Laboratory experiments were performed under controlled conditions with a simulated solar radiation specifically deprived of UV-wavebands with cut-off filters (295, 305, 320, 345 and 395 nm). The UV-insensitivity of N. flagelliforme was found to cover the whole UV-A (315–400 nm) and UV-B (280–320 nm) range and is almost certainly due to the complementary UV-absorption of MAAs and scytonemin. The experimental approach used is proposed to be suitable for the comparison of the UV-protection ability in organisms that differ in their complement of UV-sunscreen compounds. Furthermore, this study performed with a genuinely terrestrial organism points to the relevance of marine photoprotective compounds for life on Earth, especially for the colonization of terrestrial environments.     

Mycosporine-like amino acids: relevant secondary metabolites. Chemical and ecological aspects

Taxonomically diverse marine, freshwater and terrestrial organisms have evolved the capacity to synthesize, accumulate and metabolize a variety of UV-absorbing substances called mycosporine-like amino acids (MAAs) as part of an overall strategy to diminish the direct and indirect damaging effects of environmental ultraviolet radiation (UVR). Whereas the enzymatic machinery to synthesize MAAs was probably inherited from cyanobacteria ancestors via the endosymbionts hypothesis, metazoans lack this biochemical pathway, but can acquire and metabolize these compounds by trophic transference, symbiotic or bacterial association. In this review we describe the structure and physicochemical properties of MAAs, including the recently discovered compounds and the modern methods used for their isolation and identification, updating previous reviews. On this basis, we review the metabolism and distribution of this unique class of metabolites among marine organism.     

Genome Mining as an Alternative Way for Screening the Marine Organisms for Their Potential to Produce UV-Absorbing Mycosporine-like Amino Acid

Mycosporine-like amino acids (MAAs) are small molecules with robust ultraviolet (UV)-absorbing capacities and a huge potential to be used as an environmentally friendly natural sunscreen. MAAs, temperature, and light-stable compounds demonstrate powerful photoprotective capacities and the ability to capture light in the UV-A and UV-B ranges without the production of damaging free radicals. The biotechnological uses of these secondary metabolites have been often limited by the small quantities restored from natural resources, variation in MAA expression profiles, and limited success in heterologous expression systems. Overcoming these obstacles requires a better understanding of MAA biosynthesis and its regulatory processes. MAAs are produced to a certain extent via a four-enzyme pathway, including genes encoding enzymes dehydroquinate synthase, enzyme O-methyltransferase, adenosine triphosphate grasp, and a nonribosomal peptide synthetase. However, there are substantial genetic discrepancies in the MAA genetic pathway in different species, suggesting further complexity of this pathway that is yet to be fully explored. In recent years, the application of genome-mining approaches allowed the identification of biosynthetic gene clusters (BGCs) that resulted in the discovery of many new compounds from unconventional sources. This review explores the use of novel genomics tools for linking BGCs and secondary metabolites based on the available omics data, including MAAs, and evaluates the potential of using novel genome-mining tools to reveal a cryptic potential for new bioproduct screening approaches and unrevealing new MAA producers.     

Valorization of the Red Algae Gelidium sesquipedale by Extracting a Broad Spectrum of Minor Compounds Using Green Approaches

Until now, the red algae Gelidium sesquipedale has been primarily exploited for agar production, leaving an undervalued biomass. In this work, the use of eco-friendly approaches employing ultrasound-assisted extraction (UAE) and green solvents was investigated to valorize the algal minor compounds. The green methods used herein showed an attractive alternative to efficiently extract a broad spectrum of bioactive compounds in short extraction times (15 to 30 min vs. 8 h of the conventional method). Using the best UAE conditions, red seaweed extracts were characterized in terms of total phenolics (189.3 ± 11.7 mg GAE/100 g dw), flavonoids (310.7 ± 9.7 mg QE/100 g dw), mycosporine-like amino acids (MAAs) (Σ MAAs = 1271 mg/100 g dw), and phycobiliproteins (72.4 ± 0.5 mg/100 g dw). Additionally, produced algal extracts exhibited interesting antioxidant and anti-enzymatic activities for potential applications in medical and/or cosmetic products. Thus, this study provides the basis to reach a superior valorization of algal biomass by using alternative methods to extract biologically active compounds following eco-friendly approaches. Moreover, the strategies developed not only open new possibilities for the commercial use of Gelidium sesquipedale, but also for the valorization of different algae species since the techniques established can be easily adapted.     

Anti-Inflammation Activities of Mycosporine-Like Amino Acids (MAAs) in Response to UV Radiation Suggest Potential Anti-Skin Aging Activity

Certain photosynthetic marine organisms have evolved mechanisms to counteract UV-radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids (MAAs). In this study, MAAs were separated from the extracts of marine green alga Chlamydomonas hedleyi using HPLC and were identified as porphyra-334, shinorine, and mycosporine-glycine (mycosporine-Gly), based on their retention times and maximum absorption wavelengths. Furthermore, their structures were confirmed by triple quadrupole MS/MS. Their roles as UV-absorbing compounds were investigated in the human fibroblast cell line HaCaT by analyzing the expression levels of genes associated with antioxidant activity, inflammation, and skin aging in response to UV irradiation. The mycosporine-Gly extract, but not the other MAAs, had strong antioxidant activity in the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Furthermore, treatment with mycosporine-Gly resulted in a significant decrease in COX-2 mRNA levels, which are typically increased in response to inflammation in the skin, in a concentration-dependent manner. Additionally, in the presence of MAAs, the UV-suppressed genes, procollagen C proteinase enhancer (PCOLCE) and elastin, which are related to skin aging, had increased expression levels equal to those in UV-mock treated cells. Interestingly, the increased expression of involucrin after UV exposure was suppressed by treatment with the MAAs mycosporine-Gly and shinorine, but not porphyra-334. This is the first report investigating the biological activities of microalgae-derived MAAs in human cells.     

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.     

辣木氨基酸分析与营养评价研究

分析测定了辣木嫩叶、成熟叶、老叶、种子及花中的氨基酸含量,通过必需氨基酸组成、氨基酸评分、必需氨基酸指数、氨基酸比值系数及氨基酸比值系数分等5个指标对构成辣木蛋白质的氨基酸进行评价。结果表明:Glu、Asp、Lys、Leu和Arg等含量丰富,尤其是种子中的精氨酸含量分别比花生、大豆中高22.48%、40.73%。辣木嫩叶、成熟叶的氨基酸总含量及必需氨基酸含量分别极显著(p0.01)、显著(p0.05)高于老叶、种子及花,且均高于花生。辣木嫩叶、成熟叶、老叶、花在必需氨基酸含量占氨基酸总含量的比例上高于花生,且高于大豆。辣木叶、花的必需氨基酸组成中有5个项目分别高于花生、大豆、FAO/WHO模式、美国模式,辣木叶的必需氨基酸组成中有4个项目还高于鸡蛋模式。辣木叶、花中的第一限制氨基酸为Met+Cys,种子中为Lys。辣木嫩叶、成熟叶中必需氨基酸的数量充足、比例适宜,必需氨基酸组成比花生、大豆与鸡蛋中的必需氨基酸组成具有更高的拟合程度。     

L-茶氨酸对小鼠肠道形态结构及游离氨基酸的影响

将50只KM雄性小鼠按每组10只随机分为正常组、谷氨酰胺组及L-茶氨酸低、中、高剂量组,连续灌喂28 d,探究L-茶氨酸对小鼠肠道形态结构及游离氨基酸的影响。结果表明,与正常组相比,小鼠肠道绒毛高度随L-茶氨酸剂量的增大而增加,各剂量组的十二指肠绒毛高度和中、高剂量组的空肠绒毛高度及其绒毛高度与隐窝深度的比值均达显著水平(P<0.05),血清游离氨基酸含量及中、高剂量组的尿素氮含量显著降低(P<0.05),高剂量组的碱性磷酸酶、总蛋白含量显著升高(P<0.05);L-茶氨酸对不同肠道游离氨基酸含量的影响随剂量的增大而升高,其中,十二指肠中的各茶氨酸剂量组的全部必需氨基酸和大部分非必需氨基酸显著升高(P<0.05),且高剂量组的必需氨基酸含量显著高于低剂量组(P<0.05);空肠中的低剂量组的必需氨基酸Thr、Lys、Ile及中、高剂量组的全部必需氨基酸含量显著升高,且中、高剂量组中除Cit外的其他氨基酸含量均显著高于低剂量组(P<0.05);回肠中的低、中、高剂量组的全部必需氨基酸及大部分非必需氨基酸含量显著降低,且中、高剂量组中除GABA外的其他氨基酸含量显著高于低剂量组(P<0.05)。结果表明,L-茶氨酸可通过增加肠道绒毛高度、提高绒毛高度与隐窝深度比值来改善小鼠肠道形态结构,同时可促进十二指肠、空肠对氨基酸的吸收利用。     

杂交稻米必需氨基酸含量与亲本的关系

根据9个不育系、5个恢复系以及由此配制的45个不完全双列杂交组合氨基酸含量的测定结果,分析了不同组合氨基酸含量的差异及其与亲本的关系。结果表明不育系和恢复系的必需氨基酸平均含量为3.202%～4.889%,占氨基酸总量的31.796%～33.394%;而杂交组合的必需氨基酸平均含量为3.616%～4.858%,占氨基酸总量的32.176%～33.016%;必需氨基酸含量依次是亮氨酸＞缬氨酸＞异亮氨酸＞苏氨酸、赖氨酸、苯丙氨酸＞蛋氨酸。平均氨基酸总量和必需氨基酸含量都有一定的杂种优势,但各组合间差异明显。     

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.     

水稻灌浆期高温对天冬氨酸代谢酶活性及其家族氨基酸含量的影响

 选用粳稻越光和籼稻IR72为材料,研究了灌浆期高温对水稻籽粒中天冬氨酸转氨酶（AAT）、天冬氨酸激酶（AK）活性以及稻米蛋白质组分与天冬氨酸族氨基酸组分含量的影响。结果表明,水稻灌浆期高温下籽粒中AAT和AK活性、天冬氨酸、赖氨酸、苏氨酸、蛋氨酸和异亮氨酸相对含量均显著提高,籽粒中AAT和AK活性与天冬氨酸家族总氨基酸含量分别呈显著和极显著正相关,说明水稻籽粒中AAT和AK对于高温下稻米天冬氨酸家族氨基酸含量的提高具有重要作用。高温有利于稻米中蛋白质组分与天冬氨酸族氨基酸组分相对含量的提高,有利于单粒稻米中谷蛋白与天冬氨酸家族氨基酸组分的积累以及粗蛋白中部分氨基酸比例的改善,但高温降低了每穗稻米中蛋白质组分与部分氨基酸组分的含量。     

微波水解-柱前衍生-高效液相色谱法测定叶面肥中的氨基酸

建立微波水解-柱前衍生-高效液相色谱法测定叶面肥中氨基酸含量的方法。叶面肥样品在温度为160℃、微波功率为600 W下水解15 min,再与异硫氰酸苯酯进行衍生,并采用反相液相色谱法-紫外检测器对其进行分析,外标法计算样品中17种氨基酸的含量。17种阴离子在12 min内可完全分离,方法加标回收率在72.83%~98.67%之间,6次重复的相对标准偏差在3.17%~12.21%之间,方法检出限(S/N=3)在0.05 mg/L至0.15 mg/L之间。该方法操作简便、灵敏、选择性高,适用于常规样品分析。     

OPA-FMOC柱前衍生反相高效液相测定糯米香茶叶片中的氨基酸

采用邻苯二甲醛(OPA)-9-芴基甲氧基羰酰氯(FMOC)柱前衍生反相高效液相色谱法测定糯米香茶叶片中氨基酸的含量.测定了5种肥料处理下同一部位及不同部位的叶片中氨基酸含量.结果表明:5种不同肥料处理下叶片中氨基酸含量存在较大差异,其中605有机肥处理下,糯米香茶底部叶片中的氨基酸含量最高.此研究为糯米香茶的种植栽培及食品研究开发提供了科学依据.     

饲料氨基酸对褐飞虱及其蜜露游离氨基酸的影响

 利用氨基酸组成不同的全纯人工饲料对褐飞虱(Nilaparvata lugens)成虫进行饲养，研究了饲料氨基酸对该虫及其蜜露中游离氨基酸的影响。结果表明：1）饲料对试虫体内游离氨基酸有明显影响。在不能摄入任何氨基酸的试虫体内，游离氨基酸总量降至可以获得全部氨基酸的试虫的63.2%，其中缬氨酸、苏氨酸、异亮氨酸、亮氨酸、赖氨酸和半胱氨酸下降明显，但蛋氨酸增多。在仅能摄取到非必需氨基酸的试虫体内，蛋氨酸、赖氨酸和亮氨酸的含量与不能摄取到任何氨基酸的试虫处在同一水平，甚至明显低于后者；苏氨酸和组氨酸则明显高于可以获得全部氨基酸的试虫，异亮氨酸、苯丙氨酸和精氨酸亦达到了后种试虫的含量水平，缬氨酸的含量虽然低于后者，但与仅能摄取到必需氨基酸的试虫处在同一水平。2）蜜露中的游离氨基酸组成基本与饲料内的相一致，但相对含量有较大差别，而且少数不存在于饲料中的氨基酸可以在蜜露中检测到。文中对必需氨基酸的补偿代谢进行了讨论，初步认为褐飞虱体内至少可以合成苏氨酸、组氨酸、异亮氨酸、苯丙氨酸、精氨酸和缬氨酸等6种必需氨基酸。     

雄性配子诱杀剂CRMS对水稻花药蛋白质与游离氨基酸的影响

 于水稻减数分裂期施用CRMS 5～20 mg／丛可引起花药中游离氨基酸含量的大幅度下降，尤其游离脯氨酸下降更烈，进一步表明CRMS诱导水稻雄性不育的主要生理机制是氨基酸减少而引起花药蛋白质的匮乏。值得提出的是在试验中亦发现酪氨酸的严重下降，其生理意义有待进一步研究。     

播种期对杂交稻组合稻米直链淀粉和蛋白质及氨基酸含量的影响

选用湖南省洞庭湖区种植的20个杂交稻组合在9个播期下获取的180个稻谷样品,进行了直链淀粉和蛋白质含量的分析,并对杂交稻汕优63的9个播期获取的9个样品进行了17种氨基酸含量的分析.结果表明,20个杂交稻组合的9个播期的稻米直链淀粉含量平均值变幅为15.0％～24.8％,平均极差为9.8％,变异系数变幅为2.8％～21.7％;而其蛋白质含量平均值变幅为7.3％～8.6％,变异系数变幅为4.3％～11.6％.表明杂交稻组合的直链淀粉含量与蛋白质含量受播种期的影响较大,而且,其影响程度还存在明显的杂交稻组合间遗传差异.研究结果还表明,杂交稻组合汕优63的各种氨基酸的绝对含量和相对含量受播种期的影响较大,并且存在显著的氨基酸间差异.     

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。