Screening of functional components derived from fresh water laver, Prasiola japonica, and its pharmacological properties / 동물의과학연구지

The aim of the current study was to analyze the active ingredients and to screen the pharmacological properties of freshwater laver, Prasiola japonica, the only species grown in Korea. According to results of gas chromatography-mass spectrometry assay, components from P. japonica were more diverse than those from sea laver. Of particular interest, our results indicated that ethanol extract of P. japonica (PJE) contained loliolide, sorbitol, mannitol, and alverine, which were known to have an anti-oxidant, anti-oral microbial, osmotic diuresis, and smooth muscle relaxant, respectively. In addition, five solvent fractions of PJE (water, butanol, chloroform, ethyl acetate, and hexane) significantly inhibited the production of lipopolysaccharide-induced nitric oxide and a higher amount (>100 microg/mL) of chloroform, ethyl acetate, and hexane fraction were considered to play a specific role in cancer cell death. PJE and its solvent fractions found to be effective scavengers of free radicals, particularly, hydroxyl radicals. Glucose uptake in L6 myoblast cell line that stably expresses the glucose transporter type 4 (GLUT4) proteins was also remarkably enhanced upon treatment with solvent fractions, remarkably chloroform fraction. Taken together, we concluded that P. japonica may have potent pharmacological properties and thus contribute to development of novel natural candidates for various disease targets.

Antitumor function and mechanism of phycoerythrin from Porphyra haitanensis

The anti-tumor effect of R-Phycoerythrin (R-PE) from Porphyra haitanensis was studied using cell line HeLa as an in vitro model and Sarcoma-180 (S180) tumor-bearing mice as an in vivo model. The results showed that the combination treatment of R-PE and photodynamic therapy PDT) significantly inhibited the growth of HeLa cells up to 81.5%, with a fair dose-effect relationship, but did not inhibit endothelial cells. The annexin v-fitc/PI fluorescence staining experiments demonstrated that at doses between 0~60µg/mL, apoptosis cells and later stage apoptosis cells or necrosis cells increased significantly as the R-PE dosage increased. DNA electrophoresis showed that after R-PE+PDT treatment of HeLa cells for 24 hours, a light "smear" band between 100~400bp appeared to indicate the degradation of genomic DNA. The QRT-PCR results showed that R-PE+PDT treatment increased caspase-3 and caspase-10 gene expression and decreased the Bcl-2 gene expression level significantly as the R-PE dose increased, implying that R-PE promoted HeLa cell apoptosis. Compared with untreated S180 tumor-bearing mice, R-PE injection significantly inhibited the growth of S180 in tumor-bearing mice up to 41.3% at a dose of 300mg-kg-1. Simultaneously, the significant increase of superoxide dismutase (SOD) activity in serum (p < 0.01) and the decrease of the malondialdehyde (MDA) level in liver suggests that R-PE improved the anti-oxidant ability of the S180 tumor-bearing mice, which may related to its antitumor effect. In addition, the R-PE caused a significant increase (p < 0.05) in the spleen index and thymus index, and a significant increase (p < 0.01) in lymphocyte proliferation, NK cell kill activity and the TNF-α level in the serum of S180 tumor-bearing mice. These results strongly suggest that the antitumor effect of R-PE from Porphyra haitanensis functioned by increasing the immunity and antioxidant ability of S180 tumor-bearing mice, promoting apoptosis by increasing protease gene expression and TNF-α secretion.

Scale-up preparation of phycoerythrin from Porphyra haitanensis / 生物工程学报

We developed large-scale preparation of phycoerythrin from Porphyra haitanensis, a main economic red algae in China. Firstly, P. haitanensis thallus was broken by using "swelling and smash" method. Then times of grads ammonium sulfate precipitation applied to the crude extraction were compared. Desalted solution was further purified with one-step chromatography using hydroxyapatite and properties on spectrum and molecular weight were identified finally. The results indicated that after four times of ammonium sulfate precipitation (15%, 50%, 10% and 40%), the absorption spectrum purity of P. haitanensis achieved 0.9 (A564/A280), and 507.82 mg phycoerythrin (A564/A280 > 3.2) was obtained from 7 kg fresh algae after further hydroxyapatite chromatography. This research provides a potential way for preparation of phycoerythrin in large sclae.

Isolation and regeneration of transiently transformed protoplasts from gametophytic blades of the marine red alga Porphyra yezoensis

Despite the recent progress of transient gene expression systems in a red alga Porphyra yezoensis by particle bombardment, a stable transformation system has yet to establish in any marine red macrophytes. One of the reasons of the difficulty in genetic transformation in red algae is the lack of systems to select and isolate transformed cells from gametophytic blades. Thus, toward the establishment of the stable transformation system in P. yezoensis, we have developed a procedure by which transiently transformed gametophytic cells were prepared from particle bombarded-gametophytic blade as regeneratable protoplasts. Using mixture of marine bacterial enzymes, yield of protoplasts was high as reported elsewhere; however, these protoplasts did not develop. In contrast, protoplasts prepared from gametophytes treated with allantoin were normally developed, in which the overexpression of a â-glucuronidase reporter gene had no effect on the regeneration of protoplasts. Therefore, the use of allantoin in protoplast preparation sheds a new light on the realization of an efficient isolation and selection of study transformed cells from gametophytic blades.

Photodynamic effect of two kinds of phycobiliproteins on human liver cancer cell line SMMC-7721 in vitro / 生物工程学报

We studied the effect of photodynamic therapy with phycobiliproteins on human liver cancer cells in vitro. With 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), we used two phycobiliproteins, R-phycoerythrin (R-PE) and C-phycocyanin (C-PC) prepared from Porphyra yezoensis, to determine the killing rates and apoptosis rates of human liver cancer cells (SMMC-7721) mediated by laser. When the concentration of R-PE was 120 mg/L, the survival rate of human liver cancer cells was 27% after treated by Argon laser with 100 J/cm2 doses, while the survival rate in the control group (without adding R-PE) was 65%. When the C-PC concentration was 120 mg/L, the survival cell rate was 47% after treated by He-Ne laser with 35 J/cm2 dose, while the survival rate in the control group (without adding C-PC) was 70%. After handled only with these two kinds of phycobiliproteins for 72 h, the growth of cancer cells presented significant inhibition. The maximal inhibition rates reached up to 31% with R-PE (120 mg/L concentration) and 27% with C-PC (250 mg/L concentration) respectively. After irradiated by laser for 8 h, the maximal cell apoptosis rates were 31.54% with R-PE and 32.54% with C-PC, respectively. It indicated that R-PE and C-PC extracted from Porphyra yezoensis could develop to new photosensitizers for cancer photodynamic therapy.

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis.

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.