Isolation and Characterization of C-phycocyanin from Spirulina Platensis

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钝顶螺旋藻Spirulina platensis两种不同形态藻丝体的光合作用和蛋白质差异表达分析

从钝顶螺旋藻Spirulina platensis中分离纯化获得了螺旋形和直线形两种不同形态的藻丝体. 通过对二者光合作用的研究发现, 螺旋形藻丝体具有较高的光饱和光合作用速率(P_m^Chla)和光饱和点, 比直线形藻丝体更能适应较高光强的环境; 而直线形藻丝体具有较低的光补偿点, 能在更低的光强下进行光合作用. 通过双向凝胶电泳对两种不同形态藻丝体的总蛋白进行比较分析, 从中找出了9个差异表达的蛋白点. 应用基质辅助激光解吸电离质谱对差异表达的蛋白质点进行鉴定和数据库检索, 结果表明, 3个蛋白与肽聚糖代谢有关, 2个蛋白与光合作用有关, 1个蛋白与细胞分裂调控相关, 1个蛋白为外膜通道蛋白, 2个蛋白为功能未知的假想蛋白.     

Isolation and structural elucidation of a polysaccharide with radiation-protective activity from Spirulina platensis

The structure of a radiation-protective sulfated polysaccharide, SP-APS, isolated from Spirulina platensis, was investigated by methylation analysis, partial acetolysis and 1H-NMR spec-troscopy. The repeating-unit of SP-APS as branched structures was elucidated.     

The Stabilization Effect of Glutaraldehyde on the Spirulina platensis Phycobilisomes

The spectral properties of the glutaraldehyde-treated phycobilisomes were studied. The results showed that glutaraldehyde was effective in preventing phycobilisomes from dilutioninduced dissociation and preserving the intra-phycobilisomes energy transfer.     

Evaluation of antiplasmodial properties of a cyanobacterium,Spirulina platensis and its mechanism of action

The rapid emergence of antimalarial drug resistance necessitates a continual effort on novel drug discovery. A cyanobacterium, Spirulina platensis, is a potential antimalarial agent that has been widely consumed as food supplement in the form of crude extract. It is known to possess antiviral, antibacterial and antifungi activities. This study examined the antimalarial activities of several Spirulina formulas against Plasmodium falciparum 3D7, in vitro. The tested Spirulina formulas included commercially available capsule, crude extract and alkaloid fraction. Results showed that all tested formula possessed antimalarial activities with the Spirulina capsule exhibited the highest activities (IC₅₀ = 2.16 μg/mL). Light and electron microscopies revealed interference of the Spirulina with the parasite hemozoin formation. In conclusion, all tested Spirulina formulas and fraction exhibited moderate to high antimalarial activities.     

The Allelopathic Activity of Extracts and Isolated from Spirulina platensis

We determined the allelopathic effects of crude organic (hexane, ethyl acetate, and methanol) extracts of the cyanobacterial Spirulina platensis on barnyardgrass (Echinochloa crus-galli (L.) Beauv.) and Chinese amaranth (Amaranthus tricolor L.). The crude ethyl acetate extract showed the highest inhibitory activity and was subsequently fractionated by column chromatography into 23 fractions based on thin-layer chromatography band pattern similarities. Four concentrations (2000, 1000, 500, and 250 ppm) of each fraction were tested for their allelopathic activity. Fractions E6 and E13 exhibited the most significant inhibitory effects against Chinese amaranth. The constituents of the highly active E6F3-E6F5 fractions determined by GC-MS, chromatography, and spectroscopy included the fatty acids, γ-linolenic acid 15, oleic acid 12, and predominantly palmitic acid 7; minor constituents included 2-ethyl-3-methylmaleimide 9 and C₁₁ norisoprenoids (dihydroactinidiolide 10 and 4-oxo-β-ionone 13). Isolation of E13 fraction by column chromatography revealed four C₁₃ norisoprenoids: 3-hydroxy-β-ionone 17, 3-hydroxy-5α,6α-epoxy-β-ionone 18, 3-hydroxy-5β,6β-epoxy-β-ionone 19, and loliolide 20. Their structures were elucidated by NMR spectroscopy. All six isolated norisoprenoids inhibited seed germination and seedling growth of Chinese amaranth at concentrations of 250–1000 ppm. Allelochemicals from S. platensis could be utilized in the development of novel bioactive herbicides.     

富硒螺旋藻光谱特性及其含硒蛋白质组分析

以富硒螺旋藻( Selenium enriched spirulina platensis, Se-SP)为材料,采用红外、紫外-可见吸收光谱分析Se-SP的光谱特征,并利用二维电泳技术(Two-dimensional electrophoresis,2DE)分离、飞行时间质谱(Matrix assisted laser desorption ionization time of flight, MALDI-TOF)鉴定和电感耦合等离子体质谱( Inductively coupled plasma mass spectrometry, ICP-MS)检测硒含量,对Se-SP蛋白质组及其含硒蛋白质分布进行初步鉴定和分析。结果显示,硒对螺旋藻蛋白质组中多种蛋白质表达有一定影响,观察到对捕光色素蛋白如藻蓝蛋白( Phycocya-nin, PC)、别藻蓝蛋白( Allophycocyanin , APC)和 Robisco大亚基及超氧化物歧化酶( Superoxide Dismutase, SOD)等蛋白质具有明显上调作用。硒在Se-SP蛋白质组中存在较为普遍,但分布相对集中在pH 4~7、分子质量14.4~43 kDa的蛋白质中。硒在藻体特定蛋白质中的存在形式还有待深入研究和鉴定。     

Chemical composition and selenium distribution in selenium enriched Spirulina platensis biomass

Conditions for cultivating selenium enriched Spirulina platensis were developed. The protein, carbohydrate, lipids, and pigments were determined. The organic selenium and its distribution were analyzed. Published in Khimiya Prirodnykh Soedinenii, No. 6, pp. 519–522, November–December, 2006.     

Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.     

Capillary electrophoresis-mass spectrometry of Spirulina platensis proteins obtained by pressurized liquid extraction

In this work, the usefulness of CE-MS to monitor and optimize the pressurized liquid extraction (PLE) of proteins from Spirulina platensis microalga is demonstrated. Crude and purified PLE extracts from microalga were analyzed by CE-MS. It was observed that the use of purification protocols of phycobiliproteins (namely, ultrafiltration or precipitation-dialysis-freeze drying) resulted in better CE resolution and MS signals, demonstrating that sample matrix plays an important role in CE-MS of proteins in real samples. Ultrafiltration was found less laborious and much faster than precipitation-dialysis-freeze drying (1 vs. 48 h). Direct analysis of crude extracts was demonstrated to be also possible by CE-MS, providing less-quality information but enough to characterize PLE extracts in a much faster way. Therefore, the latter protocol was selected to monitor and optimize the extraction process of phycobiliproteins from S. platensis. To do that, different extraction conditions were tested, including time, temperature and pressure of extraction, nature of pressurized liquid, distribution of microalga inside the extraction cell, type of packing, etc. It is demonstrated that the combined use of PLE and CE-MS allows the attainment of extracts rich in phycobiliproteins in short extraction times (namely, yields of 20% can be obtained in less than 2 h under the optimum PLE process in an automatic way). To our knowledge, this work shows for the first time the usefulness of CE-MS for monitoring and optimizing a PLE process.     

Determination of Phenolics in Water and Arthrospira (Spirulina) platensis by Concentrated Sulfuric Acid and Ultrasound-Assisted Surfactant-Enhanced Emulsification Microextraction and High Performance Liquid Chromatography

A novel, practical, and environmentally friendly technique, termed concentrated sulfuric acid cleanup and ultrasound-assisted surfactant-enhanced emulsification microextraction (CSAC-UASEM), was combined with HPLC for the preconcentration and determination of five phenolics in water and Arthrospira (Spirulina) platensis samples. The main advantages include that the concentrated sulfuric acid is used to decrease macromolecular interferences prior to microextraction and, unlike dispersive liquid–liquid microextraction procedures, no dispersive organic solvent is required. Chloroform and sodium dodecyl sulfate were used as the extraction solvent and emulsifier, respectively. The algal cell preparation and CSAC-UASEM procedure parameters, including selection of cleanup method, ultrasound power, cell cytocylasis time, type and volume of extraction solvent, extraction temperature, ultrasound-extracted time, and sample pH, were optimized. At the fortification levels of 1.0 and 10.0 µg/L, the enrichment factors of analytes were in the range of 201.38 to 269.24. The percent extraction ranged from 71.57% to 107.42% in environmental Arthrospira-350, -793, and -834 samples, whereas the range was from 74.17% to 106.72% in water samples. The limits of detection (at S/N = 3) were 0.02 to 0.04 µg/L (except for 4-bromobisphenol A of 0.10 µg/L). These values indicate an approximately ten-fold improvement compared with the values reported by other techniques. In summary, the CSAC-UASEM sample preparation technique has great potential in the routine determination of trace phenolics in environmental waters and aquatic biological samples.     

A novel CuFe2O4@Ag nanocomposite biosynthesized by Spirulina platensis exhibits an anticancer effect on human gastric adenocarcinoma and Michigan Cancer Foundation-7 breast cancer cell lines

In recent years, the increasing cancer incidence and mortality rate has posed a significant challenge to scientists to develop novel therapeutic drugs against cancerous cells. One of the investigated techniques for cancer therapeutics is the green synthesis of nanoparticles (NPs). In this study, we reported the green synthesis and characterization of the CuFe₂O₄@Ag nanocomposite using Spirulina platensis and its cytotoxic activity on two cancer cell lines: human gastric adenocarcinoma (AGS) and Michigan Cancer Foundation-7 (MCF-7) breast cancer. The physical and chemical properties of the biosynthesized nanocomposite were characterized using Fourier-transform infrared spectroscopy, X-ray diffraction, energy-dispersive X-ray analysis, dynamic light scattering, ultraviolet–visible spectroscopy, scanning electron microscopy, transmission electron microscopy, and zeta potential analyses. The anticancer properties of the CuFe₂O₄@Ag nanocomposite and imatinib drug on both cancer cell lines were evaluated using 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Also, apoptosis induced by the nanocomposite was assessed using annexin V/propidium iodide staining followed by flow cytometry analysis, Hoechst 33432 staining, and caspase-3 activity assay. Finally, the effect of the CuFe₂O₄@Ag nanocomposite on the expression of BAX and BCL2 genes was assessed by real-time polymerase chain reaction. The result of the MTT test showed an increase in the cellular uptake of CuFe₂O₄@Ag nanocomposite and cell viability loss in a concentration-dependent manner with the 50% minimum inhibitory concentration (IC₅₀) of 180 and 220 μg/ml for MCF-7 and AGS cell lines, respectively. The mean expression of BAX was significantly higher than that of BCL2 in cells treated with the nanocomposite. The results of flow cytometry, Hoechst 33432 staining, and caspase-3 activity assay indicated the stimulation of apoptosis through an increase in caspase-3 and nucleus fragmentation. In general, our results demonstrated the cytotoxic activity of the CuFe₂O₄@Ag nanocomposite. However, further in vivo studies are required to evaluate the accumulation of this nanocomposite in organs such as liver, kidneys, brain, and testes and its potential toxic effects.     

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.