Preparation And Safety Evaluation Of Triterpene Glycosides From Sea Cucumber, Pearsonothria Graeffei

Holothurian glycosides is also called holothurin, which is a kind of triterpenoid saponins extracted from sea cucumbers. It has comprehensive physiological and pharmacological activities, but holothurian glycosides has haemolyticus strongly, which cause a big hidden danger of safety of the application of holothurian glycosides and restrict its further development of clincal application. This paper takes P. graeffei as raw material to prepare holothurian glycosides, separate and purify holothurian glycosides monomers, researches acute toxicity, mutagenicity, genetic toxicity to germ cells and 30-day toxicity of holothurian glycosides, and makes a comprehensive assessment of the safety of holothurian glycosides. The main results of this research are as follows:The preparation of holothurian glycosides. In the purification process of P. graeffei holothurian glycosides, holothurian glycosides is made by ethanol extraction, its purity can increase 85.07% by macroporous resin chromatography; its purity can further increase 40.10% by normal-phase silica gel chromatography. Holothurian glycosides monomers HA and EA are prepared by the purification of RP silica gel chromatography, and the purity of these two monomers can increase 11.71% and 4.19% respectively after separation and purification by HPLC.Holothurian glycosides has acute toxicity. The mice was gavaged with different dosage of holothurian glycosides, after 24 hours, death happened in 1g/kg and 3g/kg groups, the death exceeds half in 3g/kg group. According to the Hom method to make sure that the median lethal dose of holothurian glycosides in mice is 1.10g/kg( 1.10mg/kg for female and 1.08mg/kg for male ) .After 14 days, AST, ALT and peripheral hematological of all groups didn't have significant changes, which shows that these dosages of holothurian glycosides didn't have obvious impair to the liver of mice and with no effect peripheral blood.Holothurian glycosides has no mutagenic effect. The mice were gavaged by certain dosage of holothurian glycosides through 30 hours tested method, and the distinct of the frequency of micronuclei between holothurian glycosides groups and normal control group is not remarkable. The results show that holothurian glycosidesn without mutated actions when the gavage dosage is 138mg/kg~550 mg/kg.Holothurian glycosides has no genetic toxicity to germ cells. After gavage with different dosage of holothurian glycosides for 5 successive days, and the smears of mice sperms were observed after 35days. The results of this experiment shows that the different of abnormal sperm rate between holothurian glycosides groups and normal control group is not remarkable. Holothurian glycosidesn without genetic toxicity to germ cells actions when the gavage dosage is 138mg/kg~550 mg/kg.Rats were gavaged by different dosage of holothurian glycosides for 30 successive days, the results shows that taking holothurian glycosides for a long term can slow down the increase of body weight, without significant change of peripheral blood. Holothurian glycosides can increase the amount of leucocyte and platelet of male rats, which shows that holothurian glycosides can improve hemolysis. It can also increase the content of HDL-C and restrict the accumulation of fat, and through the tectology test, it can be found that holothurian glycosides would bring small damages to liver, spermary and ovary.This research mades to detect of acute toxicity of holothurian glycosides for the first time, and studied micronucleus, genetic chromosomal on bone marrow cells and reproductive toxicity systematically, and has an assessment of the safety of holothurian glycosides, which provide a reference of corresponding dosage for the practical application of holothurian glycosides.