Diversity and antimicrobial activity of culturable fungi associated with sea anemone Anthopleura xanthogrammica

Background: The main objective of this study was to isolate fungi associated with Anthopleura xanthogrammica and measure their antimicrobial and enzymatic activities. A total of 93 fungal strains associated with A. xanthogrammica were isolated in this study, of which 32 isolates were identified using both morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The antibacterial activities of 32 fungal isolates were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Edwardsiella tarda, Vibrio harveyi, Fusarium oxysporum, and Pyricularia oryzae by agar diffusion assay. Extracellular hydrolytic enzyme activities of the fungal isolates were determined by agar diffusion assays. Enzyme activities were detected from clear halo size. Results: The isolated fungi belonged to 18 genera within 7 taxonomic orders of 1 phylum. The genera Aspergillaceae were the most diverse and common. The antimicrobial activities of 32 isolates were evaluated, and 19 (59.4%) of fungi isolate displayed unique antimicrobial activities. All fungal strains displayed at least one enzyme activity. The most common enzyme activities in the fungi isolates were amylase and protease, while the least common were pectinase and xylanase. Conclusions: This is first report on the sea anemone-derived fungi with antimicrobial and enzyme activities. Results indicated that sea anemone is a hot spot of fungal diversity and a rich resource of bioactive natural products.

Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry

Background: Pore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17. Methods: Cytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18. Results: The amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 µg), goat (HU50 = 13.2 ± 0.3 µg), rabbit (HU50 = 34.7 ± 0.5 µg), and human (HU50 = 25.6 ± 0.6 µg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 µg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.(AU)

Study on the Antitumor Activity of Anthopleura xanthogrammica Crude Extract on SPC-A1 Cells / 中国药房

OBJECTIVE:To study the antitumor activity of Anthopleura xanthogrammica crude extract on human lung cancer SPC-A1 cells in vitro. METHODS:A. xanthogrammica crude extract obtained by the methods of repeated freezing and thawing,ac-etone precipitation. After treated with crude extract 0(blank control),0.625,1.25 and 2.5 mg/ml for 24,48 and 72 h,the activity of SPC-A1 cells were measured by MTT assay. The growth inhibition rate and IC50 were also calculated. 24 h later,the morphologi-cal changes of SPC-A1 cells were observed by HE staining and AO/EB fluorescence staining. RESULTS:MTT assay showed that A. xanthogrammica crude extract has significant inhibitory effect on the proliferation of human lung cancer SPC-A1 cells;with the increasing of the concentration and the extension of the time,the inhibitory rate was increased. Its 24 h,48 h ,72 h IC50 were 1.81,1.32 and 1.18 mg/ml. HE staining and AO/EB staining appeared obvious morphological changes of apoptosis that cell mor-phology narrowed,vacuoles arose in the cytoplasm,karyopyknosis and part of nuclear disappearance occurred. CONCLUSIONS:A. xanthogrammica crude extract has an inhibitory effect on the proliferation of human lung cancer SPC-A1 cells.

Nucleosides from Anthopleura stell / 中草药

To study the chemical constituents of sea anemone, Anthopleura stell Verrill.Methods　The chemical constituents were isolated and purified by chromatographic methods and their structures elucidated by chemical evidences and spectra data.Results　5 nucleosides were obtained and identified from the ethanolic extract as 2-hydroxy purine (Ⅰ); deoxyinosine (Ⅱ); 1-methylxanthosine (Ⅲ); deoxyguanosine (Ⅳ), and deoxyribo-thymidine (Ⅴ).Conclusion　Compound Ⅲ was a new natural product， while the others were found from sea anemone for the first time.

CHEMICAL CONSTITUENTS FROM CHINESE ANTHO-PLEURA XANTHOGRAMMICA BERKLY(II) / 中国海洋药物

From ethyl acetate fraction of Chinese Anthopleura xanthogrammica B, collected from Qingdao beach, three new glycerolipids named 1-O-hexadecanoyl-2-O (9-oc-tadecenoyl )-3-O-( 9, 12-octadecadienoyl ) glycerol (3)'1-O-hexadecahoyl-3-O-(14-eiosyle-cenoyl)glycerol (4)and l-O-(9,12-octadecadienoyl)-2-O-(9,12-octadecadienoyl)glycerol (5) as well as two lipidic acids named 9,12-octadecadienoic acidd) and 9,14-docosandienoic acid (2) were isolated. Their structures were identified by various spectrial(IR,PNMR ,CNMR, MS,et al)analysis and chemical conversion.