Extraction And Separation Of Astaxanthin From Haematococcus Pluvialis And Its Activity Evaluation

Astaxanthin possesses good application prospect due to its strong anti-oxidation,anti-aging,immune function enhancement and some other biological activities.Haematococcus pluvialis was reported to be the most abundant natural source of free astaxanthin and its esters.However,due to the physicochemical instability and the presence of its cis-isomer,astaxanthin,especially the free astaxanthin,was hard to be extracted via the traditional extraction and separation methods.The high temperature and long-time treatment in the traditional methods may lead to the oxidative degradation or the conversion of isomers of astaxanthin components.The difficulty of separating free astaxanthin lead great limitation for the study of its activity as well as the development and utilization of its products.In order to solve the problems above,two new methods for the extraction and separation of astaxanthin from Haematococcus pluvialis were carried out in this issue,including the disruption extraction with low temperature and high pressure and the high pressure supercritical CO₂ extraction.At the same time,the antioxidant activity and anti-tumor activity of the various extracts and free astaxanthin monomers of Haematococcus pluvialis were evaluated by various anti-oxidation evaluation methods and anti-tumor activity screening model in vitro.This issue provides advanced technology for the preparation of astaxanthin.Additionally,the issue also provides the theoretical and technical basis for the development of new products.The main contents and results of this study were listed as follows:1.Two compounds were isolated from Haematococcus pluvialis by supercritical CO₂ extraction associated with industrial chromatography.The two compounds were characterized using UV,LC-MS,NMR(¹H-NMR,¹³C-NMR).The results of spectral were compared with the reported data.The two compounds were identified to be trans astaxanthin and dehydro astaxanthin,respevtively.The result of this work provided a reference for the quantitative analysis of the extraction and separation process.2.The methods for the determination of total astaxanthin and main free astaxanthin was established by ultraviolet spectrophotometry and high-performance liquid chromatography.The two methods were accurate and reliable with good precision and reproducibility that can be used for the determination of total astaxanthin and main free astaxanthin.3.The disruption extraction with low temperature and high pressure and the high-pressure supercritical CO₂ extraction was designed to extract astaxanthin from Haematococcus pluvialis.Taking the yield of the Haematococcus pluvialis extract and the yields of trans-atx and dehydro-atx as the evaluation index,the optimal operation conditions of low temperature and high-pressure disruption extraction were set as follows:The solvent was ethanol,the ratio of solid to liquid was 1:30 and the extraction pressure was 150 MPa.Twice treatment was needed during the process.The extraction time was 30min.The yield of Haematococcus pluvialis extract was14.43%.The yield of total astaxanthin was 4.18%.The yields of trans-atx and dehydro-atx were 2.18%and 0.76%,respectively.The content of total astaxanthin was21.29%.The contents of trans-atx and dehydro-atx were 13.58%and 4.29%,respectively.The optimal operation conditions of high-pressure supercritical CO₂ extraction were set as follows:The extraction temperature was set as 50°C.The extraction pressure was set as 65 MPa.The separation kettle I temperature was set as 50°C.The separation kettle I pressure was set as 8 MPa.The separation kettle II temperature was set as 40°C.The separation kettle II pressure was set as 6 MPa.The extraction time was set as 30 min.The yield of extract was 18.15%.The yield of total astaxanthin was4.86%.The yields of trans-atx and dehydro-atx were 2.78%and 0.79%,respectively.The content of total astaxanthin was 28.20%.The contents of trans-atx and dehydro-atx were 15.31%and 4.35%,respectively.The yields and contents of astaxanthin extract by these two new techniques were significantly higher than those of traditional extraction methods.Among these methods,the high-pressure supercritical CO₂ extraction had dominant advantages.The kinetic study of high-pressure supercritical extraction process showed that the method with high pressure extraction?65 MPa?has more advantages than the method with traditional pressure extraction?lower than 40 MPa?.4.The industrial chromatography separation technology was used to separate and purification the monomers from the Haematococcus pluvialis extract obtained by high pressure supercritical CO₂ extraction method.Taking the relative purity as the evaluation index,the optimal operation conditions of industrial chromatograph were set as follows:C30 packing was used as chromatographic packing.The eluent was methanol with the content of 98%.The flow rate was set as 13 mL/min.The optimal injection volume was 5 mL.The detection wavelength was 478 nm.The relative purity of trans-atx and dehydro-atx were higher than 98%.The two free astaxanthin can be prepared on a large scale according to the above methods with optimum condition.5.The antioxidant activities of the extracts and free astaxanthin monomers of Haematococcus pluvialis were studied by iron reduction method,ABTS method and FRAP method.The results showed that the extracts and free astaxanthin monomers had strong antioxidant activity.The antioxidant activities of both free astaxanthin monomers were stronger than that of each extract.The trans-atx possessed the highest antioxidant activity following by dehydro-atx.At the same time,the MTT assay was used to evaluate the inhibitory effect of the extract of Haematococcus pluvialis and two free astaxanthin monomers on HepG-2tumor cells?MCF-7 tumor cells and A549 tumor cells.The results showed that trans-atx had a certain inhibitory effect on HepG-2 tumor cells with an inhibitory rate of 72.89%.Dehydro-atx has a strong inhibitory effect on HepG-2 and MCF-7 tumor cells.When the concentration was 200?g/mL,the inhibitory rate of HepG-2 tumor cells was 98.74%.The IC₅₀0 value was 140.5?g/mL.The inhibitory rate of MCF-7tumor cells was 97.13%.The IC₅₀0 value was 118.9?g/mL.In addition,the extracts of Haematococcus pluvialis and two free astaxanthin monomers had no obvious inhibitory effects on A549 tumor cells.