Extraction, Purification And Anti-tumor Activity Studying Of Cerebrosides From Asterina Pectinifera Offal

Asteroidea is widely various and rich in natural resources in China, which is adaptable andeasy to propagate. But it is a low-value food and natural enemies of breeding shellfish. Asterinapectinifera which is a kind of Asteroidea containing many biologically active substances hasbecome a hot research. Cerebrosides are a variety of highly active endogenous substances withthe effects of regulation of cell growth, anti-tumor, anti-bacterial, anti-viral, anti-ulcer,immunomodulatory, neuroprotective and so on. Nowadays, the research of the anti-tumoractivity of cerebrosides has become an international mainstream and cerebrosides are expectedto be developed into a new drug which is an auxiliary treatment of neoplastic diseases.In this paper, Asterina pectinifera offal was used as raw mateials. Cerebrosides wereextracted by the method of organic solvent extraction and separated and purified by silica gelchromatography combined with thin-layer chromatography. While the extraction method wasoptimized by high-performance liquid chromatography-evaporative light scattering detectionand the structure and anti-tumor activity of separated and purified cerebrosides. The mainresults obtained are as follows:1. Asterina pectinifera offal powder was exracted by95%ethanol at room temperature andthe crude cerebrosides were obtained by the identification of thin-layer chromatography (TCL).Then the crude cerebrosides were sepatated and purified by silica gel chromatograohy manytimes and the results of TCL displayed determined that cerebrosides were purified.2. The results of structural analysis of the separated and purified cerebrosides by infraredspectrum were as follows: C-H, C=O and N-H indicated the amide bond, C-O,-CH2-andlong-chain fatty. By high resolution mass spectrometry analysis, the possible relative molecularmass were790.6439,804.6601and818.6752, corresponding formula C44H87O10N，C45H89O10Nand C46H91O10N.3. Extraction factors of cerebrosides in Asterina pectinifera offal were optimized by singlefactor experiment and orthogonal experiment. The optimal conditions were as follows: the ratioof hexane and ethanol was2:1(V/V), solid-liquid ratio was1:20(W/V), extraction time was3days and extraction times were2times. Under these conditions, the relative extraction rate ofcereborsides from Asterina pectinifera offal can reach87.67%.4. Cerebrosides were conducted anti-tumor activity in vitro experiments. From the resultsof MTT assay, cerebrosides showed cytotoxic activity against human hepatoma cells(BEL7402), human cervical carcinoma cells (HeLa) and human breast cancer cells (MCF-7)and cytotoxicity of cerebrosides on three human tumor cells showed a dose-andtime-dependent manner basically at the concentration range of10~400μg/ml. The test results of lactate dehydrogenase(LDH) in human breast cancer cells (MCF-7) culture fluid were basicallyconsistent with the results of MTT assay, and the larger dose of cerebrosides were, the moreLDH in cells leaked and the higher the activity of LDH was. It indicated that cerebrosidesincreased the membrane permeability of MCF-7cells effectively.5. Anti-tumor mechanism of cerebrosides was studied from the perspective of apootosis.Through the morphological observation(inverted microscope and fluorescent staining),cerebrosides decreased the number of MCF-7cells, rounded cell shape and decreased celldiopter, accompaning apoptotic features about chromatin shrinked, nuclei showed a dense stainand apoptotic bodies. Flow cytometry analysis showed that cerebrosides could induce MCF-7cells apoptosis and their cell cycle was arrested in the G2/M phase. According to the results ofmitochondrial transmembrane potential, cerebrosides deceased mitochondrial transmembranepotential of MCF-7cells, indicating that cerebrosides inducing MCF-7cells apoptosis wasachieved by regulating the permeability of mitochondria.