Hideomi Amano

Isolation of a new anti-allergic phlorotannin, phlorofucofuroeckol-B, from an edible brown alga, Eisenia arborea.

Eisenia arborea is an edible brown alga occasionally used as a folk medicine in gynecopathy in Japan. A new phlorotannin was isolated from the alga during our search for naturally occurring anti-allergic compounds from edible algae guided by the inhibitory effect on histamine release from rat basophile leukemia (RBL)-2H3 cells. The phlorotannin was called “phlorofucofuroeckol-B.” Its structure was determined by spectral analysis and chemical conversion. This paper describes the isolation, structure elucidation, and inhibitory effect of phlorofucofuroeckol-B on histamine release.

Antitumor activity of marine algae

Powdered tissue from 46 species of air-dried marine algae (four green, 21 brown and 21 red algae) were screened for antitumor activity. Significant activity against Ehrlich carcinoma was found in the brown algae Scytosiphon lomentaria (69.8% inhibition), Lessonia nigrescens (60.0%), Laminaria japonica (57.6%), Sargassum ringgoldianum (46.5%), the red algae Porphyra yezoensis (53.2%) and Eucheuma gelatinae (52.1%) and the green alga Enteromorpha prolifera (51.7%). Five brown and four red algae showed appreciable antitumor activity against Meth-A fibrosarcoma. To identify specific molecules with antitumor activity, 15 kinds of polysaccharide preparations of seaweed origin and 24 kinds of lipid fractions extracted from various seaweeds were tested. Appreciable inhibition of Ehrlich carcinoma was found for fucoidan preparations from Undaria pinnatifida and Sargassum ringgoldianum, for carrageenans and for porphyran. Several glycolipid and phospholipid fractions from brown and red algae were effective against Meth-A fibrosarcoma.

Effect of a seaweed mixture on serum lipid level and platelet aggregation in rats

To assess the effect of a seaweed mixture on lipid levels in serum as well as platelet aggregation in rats, Eisenia bicyclis (‘Arame’), Hizikia fusiformis (‘Hijiki’) and Undaria pinnatifida sporophylls (‘Mekabu’), all brown seaweeds, and Porphyra yezoensis (‘Susabinori’), a red seaweed, were powdered and mixed in a ratio of 45:30:20:5 (w/w). When rats were fed a cholesterol-rich diet containing this mixture of seaweeds (9–10% w/w) for 28 days, serum total cholesterol, LDL-cholesterol, free cholesterol, and triglyceride levels declined significantly to 49.7%, 48.1%, 49.0% and 74.8%, respectively, of those of the control. Serum HDL-cholesterol, however, was unchanged. Though activated partial thromboplatin time, prothrombin time, antithrombin III activity, and fibrinogen levels in plasma were unchanged, the maximal ADP- and collagen-induced platelet aggregation decreased significantly to 89.0% and 85.5% control levels, respectively. These results indicate that this mixture of E. bicyclis, H. fusiformis, U. pinnatifida sporophylls, and P. yezoensis, is useful for the prevention of hyperlipidemia and thrombosis in rats.

Inhibitory effects of seaweeds on histamine release from rat basophile leukemia cells (RBL-2H3)

The effects of 80% methanol extracts from frozen samples of 41 macroalgae and one sea grass collected in the Ise-Shima region of Japan were investigated on histamine release from rat basophile leukemia cells (RBL-2H3) sensitized with antidinitrophenyl (DNP) IgE and stimulated with DNP-BSA. Of the 21 brown algae, five green and 15 red algae, and one sea grass tested, only extracts from seven brown algae suppressed histamine release from RBL cells, as determined by highperformance liquid chromatography (HPLC) analysis. When the cytotoxic effects of the seven brown algal extracts were investigated by Trypan blue staining, only Eisenia arborea and Sargassum thunbergil did not show cytotoxic effects. Therefore, we conclude that E. arborea and S. thunbergii may contain compounds that have antiallergic effects without inducing cell death.

Role of free d- and l-alanine in the Japanese mitten crab Eriocheir japonicus to intracellular osmoregulation during downstream spawning migration

Abstract Changes of d - and l -alanine and other osmolytes were examined in the tissues and hemolymph of the Japanese mitten crab Eriocheir japonicus captured during the maturation in the river and during the course of spawning migration downstream. Only d - and l -alanine and inorganic ions increased significantly in muscle during the maturation in freshwater from July to October and during downstream migration to estuaries in October. Inorganic ions in muscle was the highest in estuaries. In the crabs captured in the sea, glycine and d - and l -alanine were largely elevated in place of the inorganic ions declined. No other free amino acid increased in the muscle of the sea specimens. In hemolymph, total amino acids comprised only 4-10% of total osmolytes and decreased with maturation and during migration. Hemolymph inorganic ions were the highest also in estuaries. These data suggest that, in addition to a role as osmolyte, d - and l- alanine in the tissues of this strong hyperosmoregulator play an important role in the adjustment of salinity tolerance prior to and during downstream migration toward the sea.

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.

Forecasting infections of the red rot disease on Porphyra yezoensis Ueda (Rhodophyta) cultivation farms

Pythium porphyrae is a fungal pathogen responsible for red rot disease of the seaweed Porphyra (Rhodophyta). Infection forecasts of Porphyra by P. porphyrae were estimated from the epidemiological observations of Porphyra thalli and numbers of zoospore of P. porphyrae in laboratory and cultivation areas. Four features of forecasting infections were determined by relating zoospore concentrations to the incidence of thallus infection; infection (in more than 1000 zoospores L−1), microscopic infection [less than 2 mm in diameter of lesion (in from 2000 to 3000 zoospores L−1)], macroscopic infection [more than 2 mm in diameter of lesion (in from 3000 to 4000 zoospores L−1), and thallus disintegration (in more than 4000 zoospores L−1). High zoospore concentrations led to more infection. The tendency that zoospore concentration of P. porphyrae increased with the rate of infection of Porphyra thalli was generally observed in forecasting infections in both the laboratory and in cultivation areas. Based on the Porphyra cultivation areas, the accuracy and consistency of forecasting infections suggest that this method could be employed to manage and control red rot disease.

Immunological detection of the fungal parasite, Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.

Anti-allergic effects of the brown alga Eisenia arborea on Brown Norway rats

To investigate the anti-allergic effects of the brown alga Eisenia arborea. A strain of Brown Norway rats know to strongly respond to immunoglobulin E (IgE) were used as an allergy model animal. The rats were immunized with ovalbumin by oral administration. The levels of serum IgE and histamine were suppressed in the rats fed a diet supplemented with dried E. arborea powder. As for the cytokine pattern, the interferon-γ production in the spleens and mesenteric lymph nodes (MLN) was enhanced, and the interleukin-4 (IL-4) production in the spleens and/or IL-10 production in the spleens and MLN were suppressed. These results, together with the change in the Th1/Th2 balance, indicate that the rats fed with E. arborea became more anti-allergic, suggesting that E. arborea might possess anti-allergic effects.

Digestion of seaweeds by the marine amoeba Trichosphaerium

A crude enzyme preparation from the marine amoeba Trichosphaerium was used to produce protoplasts from Sargassum muticum, Macrocystis pyrifera Porphyra perforata, and other red and brown marcroalgae. Cortical and medullary protoplasts of Sargassum, which were impossible to obtain using mixtures of previously available enzymes have now been prepared. Intact inner cortical and medullary protoplasts of Macrocystis, which were not observed in past isolations were obtained. Improved protoplast yields of as much as 500 fold resulted from feeding the amoebae on specific seaweeds. Cuticles of live Sargassum and Macrocystis were digested easily by the amoebae. However cuticles of autoclaved Macrocystis and those of Porphyra (fresh or autoclaved) were eaten last. In spite of the absence of identifiable extracellular enzymatic activity in the medium the amoebae were able to ‘cut’ and consume live fronds and blocks of gelled agars carrageenans, and alginates.