博士 / 國立臺灣海洋大學 / 食品科學系 / 103 / The crown-of-thorns starfish (Acanthaster planci) is the venomous starfish as well as a destroyer of coral reefs. The crown-of-thorns starfish A. planci destroys coral reefs and has been involved in significant events, such as the crown-of-thorns starfish abnormal outbreaks. The back of A. planci is covered with many sharp spines. Previous studies showed that the crude toxin extracted from the spines exhibits the following diverse biological activities: mouse lethality, hemolytic activity, capillary permeability increasing activity, edema-forming activity, phospholipase A2 (PLA2) activity and histamine releasing activity from mast cells.
In this study, the active substance extracts were done with PBS buffer. To assay the cytotoxic, hemolytic, and antioxidant properties of the water soluble extract from spine, body wall, pyloric cecum, and gonad of A. planci. Relative to other parts extract, the spine extract possessed the most biological activity, especially in cytotoxic activity. A. planci spine venom (ASV) caused hemolysis and cytotoxicity at a dose-dependent significantly. The highest activity of ASV was measured at pH 7.0-7.4; ASV-dependent hemolysis was sharply reduced when the pH was lower than 3.0 or greater than 8.0. There was almost no hemolytic activity when the concentration of Cu2+ was increased to 10 mM. Furthermore, incubation at 100 °C for 60 min sharply decreased the hemolytic activity of ASV. The cytotoxicity of ASV to human melanoma cell A375.S2 was relatively well retained at temperature less than 40°C, and sharply lost at temperature more than 80°C. The cytotoxicity of ASV also sharply lost at extreme pH environments (pH was lower than 2.0 and higher than 12.0). The cytotoxicity of ASV was attenuated when treated with Cu2+ and anti-oxidant N-acetylcysteine.
In order to understand the antitumor activity of toxin from A. planci in Penghu, Taiwan, the cytotoxic toxin of A. planci venom (CAV) was purified by fast protein liquid chromatography (FPLC) and identified as plancitoxin I. The results indicated that cells after incubating with A. planci spine venom (ASV) and purified toxin (CAV) significantly decreased cell viability and lactate dehydrogenase (LDH) level in a concentration-dependent manner was increased. The assays indicated that purified toxin promoted reactive oxygen species (ROS) productions, loss of mitochondrial membrane potential (ΔΨm), and inter-nucleosomal DNA fragmentation in A375.S2 cells. Following, the study was investigated the molecular mechanism underlying the cytotoxicity function of palncitoxin I by focusing on the oxidative stress, mitochondrial dysfunction and endoplasmic reticulum (ER) stress pathway in human melanoma A375.S2 cells. The CAV was found to reduce the cellular antioxidant enzymes such as SOD and CAT, and there was significantly decreased in total thiols level and mtDNA integrity, and enhanced the lipid peroxidation. In addition, CAV increased cytosolic Ca2+ concentration, and enhanced the expression of the ER molecular chaperones GRP78 and CHOP in a dose-dependent manner. CAV significantly elevated the activity of caspase-3, -8 and -9, and reduced the ratio of Bcl-2/Bax. The results demonstrated that plancitoxin I inhibits the proliferation of A375.S2 cells through induction of oxidative stress, mitochondrial dysfunction and ER stress associated apoptosis.
Many studies currently researching marine invertebrates to determine the therapeutic potential of their bioactive materials have been showing very promising results. Starfish possesses many useful pharmacological and biological characteristics. In this study, A. planci was extracted with 70% ethanol and lyophilized to obtain an ethanol fraction. The ethanol fraction was dissolved with water and defatted with petroleum ether to obtain a non-polar fraction. The residual solution was successively partitioned with ethylacetate and butanol to obtain an ethylacetate fraction and butanol fraction, respectively. Four fractions were used to examine the antioxidant and anticancer properties. The ethanol fraction of A. planci contained the highest antioxidant effects such as ABTS, DPPH, Fe2+ chelating activity and reducing power when compared with four fractions. Among the four fractions, the butanol fraction was especially shown to inhibit human malignant melanoma A375.S2 cells’ proliferation, which is involved in the apoptotic progression. This fraction could induce apoptosis and even necrosis in A375.S2 cells as evidenced by double staining with an annexin V-FITC and PI assay and DNA fragmentation analysis. These results indicated that the starfish A. planci is a good resource for obtaining the biologically active substances for antioxidant and anticancer effects.
However, there was little toxicological information of asterosaponins. The study evaluated potential toxicity of the asterosaponins and analyzed the oxidative stress harm in mice. The present work evaluated the toxicity of the butanol fraction (BF) of crown-of-thorns starfish (Acanthaster planci) in ICR mice, and the BF was used as the asterosaponins extract sample. In acute toxicity assay, the LD50 of the BF is about 520.34 mg/kg body weight. In sub-acute toxicity test for 28 days, the BF significantly increases in hematological parameters, including red blood cell, hemoglobin, hematocrit and mean corpuscular hemoglobin concentration, it also causes hemolytic activities. The BF induces the hepatotoxic injury through induction of oxidative stress by elevating the lipid peroxidation and decreasing the antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and total thiols. Based on microscopic observations, the BF showed harmful effect on the histomorphology change of liver. Thus, the bioactive secondary metabolites in the BF of A. planci induce a harmful effect in ICR mice.
| Identifer | oai:union.ndltd.org:TW/103NTOU5253004 |
| Date | January 2015 |
| Creators | Lee, Chi-Chiu, 李志昭 |
| Contributors | Hwang, Deng-Fwu, 黃登福 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 157 |
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