The Study Of Extraction-purification Process And Biological Activity In Acaudina Molpadioides Total Saponins

Ocean is a huge natural medicine database which contains rich natural resources. Humanbeings is facing the serious challenges of all kinds of complicated illnesses, stubborn disease,and the land medicinal resources can not satisfy people’s increased demand. Thus marineprecious resources are undoubtedly the most popular choice for human. Many countries have paymore and more attention to the research and development of marine natural products. China hasa natural advantage since it has vast waters, abundant marine products, and a long history ofapplication of marine medicine. To own one seat at the international stage, developing oceanblue drugs is an unquestionably important strategy for China. As a kind of food and medicine,sea cucumber has extremely high health care function and medicinal value. The sea cucumbersaponin is a kind of sea cucumber secondary metabolites, which has a broad range of physicalpharmacological activities, hiding great application value. This paper focus on AcaudinaMolpadioides in which the total saponins had been researched on extraction and purificationprocess, and part of the pharmacological activities, which could provide the theoretical basis forcomprehensive exploitation and utilization of Acaudina Molpadioides.1) Analysis on the basic elements of Acaudina Molpadioides body wall, that of the results:the contents of moisture, protein, total sugar, crude fats and ashes were89.64%,7.93%,0.22%,0.02%and1.76%respectively, thereinto, the protein content convert into dry weight reached76.54%; Amino acid composition totaled8.22%, eliminated the amino acid composed crudeprotein, it still had0.29%of free amino acids, which had highest content of glycine, least contentof methionine. The essential amino acids were7kinds, and total content was1.96%,non-essential amino acid were9kinds, and total content was6.26%. The amino acidcomposition was more integrated, explaining that Acaudina Molpadioides was a kind ofhigh-protein and low-fats sea food that possessed high nutritional value; In addition, with theginseng saponins Re was as standard reference, and vanillin-glacial acetic acid-high chlorineacid was taken as color-forming reagents, there was a method of the total saponins contents hadbeen determined. The spectral scanning receive the best wavelength was537nm. Linearregression equation was y=0.0038x+0.0071, r=0.9996, meant that concentration had a goodrelationship with the absorbance when it was among40-200μg/mL. The average recovery was 101.9%, RSD=4.63%. Precision experiments RSD=1.77%, stability experiments RSD=3.16%,repetitive experiments RSD=3.15%, explained that the indexes of methodology met therequirements. With application of this method to analyze the different extraction methods andformer treatment on Acaudina Molpadioides total saponins, the results showed that alcoholextraction holded the best effect, fresh sea cucumber saponins content was0.036%, while thefreeze-drying processing sea cucumber had the highest yield of0.514%after ethanol-extraction.2) Optimizing the extraction process of the total saponins from Acaudina Molpadioidesbody wall, which were fresh and extracted by alcohol. The results of single factor experiment ofextraction technology were followed: alcohol chroma was70%, extraction temperature was70°C, time was3h, and extracting3times, solid-liquid ratio was1:2.0g/mL. The results of theresponse surface analysis were concluded: alcohol chroma was75%, and extracting165min for3times at73.5°C, solid-liquid ratio was1:1.8g/mL. In this condition the experiment wasrepeated three times and measured the average that saponins yield was0.0378%.3) Purification process of the Acaudina Molpadioides total saponins was discussed.compared the three models resin （D101, AB-8, DM130） which was the best one for separationand purification of the saponins coarse liquid, the D101macroporous resin was screened for themost suitable resin type. Studying on the static adsorption-elution technology of D101macroporous resin on the coarse extraction fluid, the best condition was obtained: Taking theD101macroporous resin1.0g that had been treated by vacuum filtering, adding4.0mg/mLsample liquid60mL, then putting it on the constant temperature shaker（25°C,120r/min） andincubating90min, filtering, the resin was rinsed repeatedly with distilled water, vacuum suctionfilter, and adding30%ethanol to remove impurities, filtering, finally eluting with80%ethanol60mL, putting it on the constant temperature shaker（25°C,120r/min） and adsorpting120min.The income saponins purity was about51.4%. The dynamic adsorption-elution technology ofD101macroporous resin on the coarse extraction fluid was also investigated, the best conditionswas concluded: Taking the concentration was3.6mg/mL of saponins coarse extraction liquid onthe160mL column, the sample velocity was2.5mL/min, then used140mL （3BV） todeionizedwater to remove the water-soluble impurities,140mL （3BV）30%ethanol solution to removesome other impurities, finally used150mL （about3BV）90%ethanol solvent to elute the totalsaponins, the eluting velocity was1.5mL/min. The income saponins purity was reached71.3%.4) Studying on the hemolytic test and acute toxicological test of the total saponins, theresults were concluded: Acaudina Molpadioide total saponins holded its strong ability tohemolysis, and when the final concentration was2.5μg/mL, it began to appear hemolysis, andwith the increase of concentration, the rate rise dramatically. The hemolysis had mainlyhappened during60min, showed that the hemolysis was quick. Mice acute toxicological testing come out that the maximum tolerance dose was8000mg·kg-1. In this doses oral taking the totalsaponins was relatively safe. The test showed that the Acaudina Molpadioides total saponinswere not suitable for clinical injection, oral use would be comparative safety.5) Investigating the Acaudina Molpadioides total saponins on the antioxidative activity, theresults were described as follows: Although the total saponins presented very weak ability ofclearing O₂·–,·OH and deoxidization, which showed good effect on the mice liver homogenatelipid peroxidation inhibition. The IC₅₀was20.50mg/mL for MDA produced by the mice liverhomogenate spontaneous lipid peroxidation while9.37mg/mL for Fe²⁺-H₂O₂inducted mice liverhomogenate lipidperoxidation produced MDA. In conclusion, the main function of AcaudinaMolpadioides total saponins was not on the oxidation.6) The kunming mice was used on animal experiments, studing the effect of AcaudinaMolpadioides total saponins on resisting fatigue, hypoxia, depressants from the mice. the resultswere showed as followed: middle, high dose of saponins (160,320mg·d^-1·kg^-1) holded thesignificantly effect of reducing weight of mice, and extending weight loading swimming time ofmice; High doses could effectively increase the mobilization of energy material when anaerobicrespiration, and elevate the blood lactic acid amount; Middle, high dose could accelerate thespeed of removing lactic acid significantly at mice sport recovering moment; The total saponinscould not reduce blood urea generation; Middle, high dose of saponins did not increase the liverglycogen storage, but had an adverse effect, high dose could increase muscle glycogen reserves;High dose could also effectively relieve mice anoxic suffocative time, and reduce the miceswimming fixed time; Middle, high dose could reduce the tail suspension fixed time of mice. Allthe above explained the total saponins could obviously improve the body sports endurance,effectively prevent the accumulation of blood lactic acid, increase muscle glycogen storagefunction, resist fatigue production; And to antihypoxia, antidepressants it also had exact role.Therefore the Acaudina Molpadioides total saponins had greatly potential economic value todevelop into biological health products.This subject was committed to the development and utilization of the low value AcaudinaMolpadioides, which emphasized that it had very precious nutrition value, and was worthdeveloping.