Interaction Of Marine Natural Active Ingredients To The Cells

Screening active lead compounds with medical values from marine organisms is supposed to be an important trend in the field of research and development of drugs, first step of which is to distinguish the active components. Traditional medicine screen relies on antibacterial tests, cell model in vitro and animal model to investigate the physiological effects,which is a complicate,demanding and time-consuming process. This paper provides a new approach to screen active components in marine organisms according to the possible mechanisms between active components and cancer cells.First of all,establishing methods on activity analysis and measurement taking the known traditional Chinese medicine(TCM) as a model. Qualitative and quantitative analysis was conducted by HPLC to investigate HSYA in red flower water extracts and tetrahydropalmatine in Corydalis tuber water extract. Furthermore, effects of HSUA and tetrahydropalmatine on viabilities of liver cancer cell HepG2 were studied via CCK-8 method, and the results were in line with previous studies, which proved that the established method may be applied in the separation and activity tests of naturally active compounds.Secondly,studying 13 kinds of active compounds from extracts of natural marine organisms by both HPLC-RID and HPLC-MS. Taking ultrapure water and acetonitrile as flow phase,extracts of native marine organisms were separated by RID detector and isocratic elution program. Results showed obviously detected peaks in 5 samples, among which GG07 and KG04 had a good separation effect; Taking ultrapure water and methanol as flow phase,extracts of native marine organisms were analyzed by MS, and polysaccharides were inferred to exist in extracts considering results from both HPLC and MS.On this basis, HPLC-MS was adopted to analyze potential interaction between Escherichia coli and Staphylococcus aureus and some certain components in the sample KG04 from chitosan, and the sample HJ12 from spongia extracts. It was indicated that a component with a molecular weight of 531 kD in KG04 and a component with a molecular weight of 403 kD in sample HJ12 had low chances to be interacted with both E. coli and S. aureus, and no obvious changes were observed as the concentrations of samples increased. Besides, the interaction between HepG2 cells and two key components in sample HJ12, with the molecular weights of 401 and 403 kD, respectively, had been studied. Binding rates between HepG2 cells and these two components increased as the concentrations increased. Consequently, sample HJ12 from spongia extracts may be related to some significant physiological functions, such as inducing the apoptosis or death of cancer.Moreover, antibacterial tests and antineoplastic tests were both performed to confirm the relationships between active compounds from marine organisms and microbes and cancer cells. In antibacterial tests, we found 12 kinds of marine natural products to be lack of obvious antibacterial activities, which showed no inhibition zones after 24 h of interaction with both E. coli and S. aureus. We also explored the effects of two components from spongia extracts, with the molecular weights of 403 and 431 kD, respectively, on activities of HepG2 cancer cells by CCK-8 method and flow cytometer(Annexin V-FTIC/PI). We found that rates of apoptosis of HepG2 cells went up with the increase of concentrations of spongia water extracts. Apoptosis in the early stage was quite consistent with that in the late stage, which indicated that these two active components might induce cancer cells to unlock the process of apoptosis. Compared with the binding rates in Chapter 3, the higher rates between active components and cancer cells are, the more obvious induction of apoptosis is.Considering all results achieved above, antineoplastic activities of extracts from some other marine organisms were studied, and results were listed as below:Sample QJ03 from oligosaccharide in red algae, sample YM06 from oligosaccharide in isomalt, sample SS10 and SM11 from extracts of coral were all investigated. For the present experimental conditions, no changes were observed between normal cells and apoptosis cells, and rates of apoptosis cells kept a stably low lever without apparent proliferation, which showed the tiny effects of samples QJ03, YM06, SS10 and SM11 on cells. Sample KL02 from oligosaccharide in red algae made rates of apoptosis cells to increase, followed by an obvious decrease in the end, but the reasons to this phenomenon still needs to be work out in the future. Fortunately, cell apoptosis rates increased with the action of sample HZ01 from oligosaccharide in brown algae, sample KG04 from oligochitosan, sample MG05 from xylo-oligosaccharide, sample GG07 from fructo-oligosaccharide, samples SB08 and CC09 from sea house extracts, samples HJ12 from spongia extracts and spongia water extracts. Rates of induced apoptosis did not further increase with the concentrations increasing for HZ01, MG05, GG07 and CC09, but for SB08, no obvious changes were found in induced apoptosis rates at low concentrations, and whereas enhanced induction of apoptosis rates existed at high concentrations. On the contrary, induced apoptosis rates of KG04 increased as the concentrations of sample increased, which may have wide applications in the future.Novelties in this study focus on establishing the method of fingerprint analysis of interaction in vitro between active components in marine organisms and cancer cells by HPLC-MS, and on proposing the opinion that binding rates between active components and microbes and cancer cells are quite consistent with antibacterial activities and antineoplastic activities of marine organisms. Binding rates that are calculated by the interactions between active components and different cells, are supposed to be an important representation of the antineoplastic activities of the active components, which may eventually provide a fast and simple approach for medicine screen from marine organisms.