| Hippocampus,a marine teleost fish,is one of the valuable medicine in Asian countries including China,but also the characteristic resources of the tropical region Hainan.Hippocampus is rich in physiological active materials and it has been studied for its diverse biological activities,thus various pharmacological effects are found,such as anti-tumor,anti-oxidation,anti-aging,and so on.In this experiment,we extracted the small molecular alcohol extracts from the three-spot hippocampus.Petroleum ether extract was obtained by systematic solvent extraction from ethanol reflux extracts.The anti-inflammatory active components were obtained by silica gel column chromatography eluting by solvents with polarity divergence,and the amount of inflammatory mediator and cytokines was inhibited in murine macrophages RAW264.7 in vitro.Glycosphingolipids and long chain bases were prepared from glycolipids,the antitumor activities upon human hepatocellular carcinoma cells HepG2 and human chronic myeloid leukemia cells K562 were detected.The aqueous extract was subjected to column chromatography with the dextran gel G-10 and the antioxidant activitives of the different fractions were determined.This study is intended to provide refernce and basis for further study of physiological activity to improve the functional food and medicinal value of three-spot seahorse.In this paper,the main research contents and results are as follows:1.Isolation and evaluation of anti-inflammatory activity in vitro of active components of three-spot hippocampusIn this paper,a model of cell inflammatory responses model of monocytes macrophages RAW264.7 in vitro activated by lipopolysaccharide was established,and inhibitory capacity of the production of secretary type inflammatory mediator nitric oxide in the supernant of the cultured media was regarded as an screening index of anti-inflammatory activity of seahorse.Three-spot seahorse was the raw materials,it was sreened through 20 mesh sieve after crashed,analytical reagent 95%ethanol was used for reflux extraction to obtain alcohol extracts,and the fractions with different polarity were separated by systematic solvent extraction.Amongst the fractions,petroleum ether extract was divided into lipid soluble component,and was eluted with petroleum ether and ethanol by silica gel column chromatography.The lipids of FrIV were obtained by water washing and salt washing.Neutral lipids,glycolipids and phospholipids were prepared by using polar difference eluting with chloroform,acetone and methanol respectively by silica gel chromatography.By the preliminary judgment of thin layer chromatography and Fourier infrared spectroscopic analysis,neutral lipids were identified to be glycerol esters,the glycolipids were identified to be contain glycosphingolipids and glycoglycerolipids,phospholipids may be composed of phosphatidyl ethanolamine and phosphatidylcholine.A comparison between the anti-inflammatory activities of different lipids was made in the present study.The cell viability was detected via MTT method,ELISA assay was used to determine the contents of inflammatory cytokines in supernatant of cultured media.The results of MTT test indicated that fractions had no obvious cytotoxicity on the cells at the concentration up to 50?g/mL,and inhibited the production of nitric oxide in lipopolysaccharide stimulated murine macrophages RAW264.7 in vitro.Lipids from seahorse showed a positive correlation with the in vitro suppression of the release of TNF-a,IL-1?,IL-6.The inhibition rates of glycolipids at the concentration of 50 ?g/mL upon the production of NO,TNF-?,IL-1?,IL-6 in lipopolysaccharide induced mouse macrophages RAW264.7 in vitro were(98.85± 5.29)%,(91.74 ±1.42)%,(77.13 ±3.40)%,(93.82±1.07)%respectively.And the anti-inflammatory activities of glycolipids may have association with the structure of fatty acids,saccharide grous and sphingolipid bases.2.Isolation and evaluation of anti-tumor activity in vitro of active components of three-spot hippocampusIn the present study,the antitumor activities of glycosphingolipids and long chain bases were investigated.The crude long chain bases were purified by silica gel column chromatography to elute other polar components,and macroporous resin HP-20 was used to remove impurities.Antitumor activities were measured via MTT assay and were in dose and time dependent manner.IC50 values of long chain bases upon suppression of HepG2 cells were 8.87,6.42 ?pg/mL respectively at the interval of 24 h,48 h;the IC50 values of long chain bases upon inhibition of K562 cells were 35.54,23.18 ?g/mL respectively at the interval of 24 h,48 h.Long chain bases exerted significant anti-tumor activities compared with glycosphingolipids,which indicated that the long chain bases might be the main active structure for the anti-tumor activities of glycosphingolipids.3.Isolation and evaluation of anti-oxidative activity of active components of three-spot hippocampusIn the present study,the antioxidant activities of aqueous extract from three-spot seahorse were studied,six fractions were obtained by eluting through Sephadex G-10 gel filtration chromatography of aqueous extract of seahorse.Anti-oxidative activities of DPPH free radical scavenging,and hydroxyl radical scavenging,total reducing power and ferrous ion chelating ability were determined and evaluated with multiple concentrations and total polyphenols and total amino acids of varying fractions.The results indicated that by contrast,DPPH free radical clearance of FrIV was the highest with the value of IC50 was 0.25 mg/mL.Total reducing power FrIV was potent,FrIV exhibited the highest capacity for scavenging hydroxyl radical and ferrous ion chelating ability with values of IC50 were 1.32 mg/mL,27.41 mg/mL respectively.Collectively,the antioxidant activity of aqueous soluble extract with small molecules of three-spot hippocampus seemingly to have correlation with the distinct contents and types of phenolic compounds,amino acids,small molecular peptides. |
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