Optimization Of Culture Conditions Of Haematococcus Pluvialis And The Effect Of Its Product Astaxanthin On Gestational Diabetes

Haematococcus pluvialis is currently considered to be the most suitable microalgae for large-scale production of astaxanthin,mainly cultivated in Yunnan Province of China.The Chenghai area of Lijiang is one of the main H.pluvialis cultivation bases.The water quality environment is more alkaline and high hardness,which is very beneficial to the cultivation of Spirulina,but not good for the growth of H.pluvialis,becoming the factors limiting the large-scale cultivation of H.pluvialis.In this study,sodium alginate was used to soften the hard water of H.pluvialis cultivation in Chenghai area,and explored the influence of softened water on the cultivation of H.pluvialis.5%sodium alginate was used to soften the hard water of H.pluvialis culture,and the influence of sodium alginate dosage and action time on the adsorption of Ca²⁺and Mg²⁺in hard water was explored.Then,the effects of culture hard water and sodium alginate softened water on the growth of H.pluvialis,chlorophyll a,chlorophyll fluorescence parameters,dry weight of algae cells and astaxanthin accumulation were compared.It was found that when the weight ratio of treated water to sodium alginate was 200:1 and the adsorption time was at least 12h,Ca²⁺and Mg²⁺in the water were reduced by 55%and 39%,respectively.Compared with the hard water group,the sodium alginate softened water culture H.pluvialis increased the density of algae cells by 2 times,the content of chlorophyll a increased by 2.04 times,and the fluorescence parameters of chlorophyll showed a slight increase.In addition,after the algae cell culture was completed,the dry weight of algae cells in the softened water was 1.94 times that of the hard water group,and the astaxanthin content was 2.52 times that of the hard water group.The experimental results show that the use of sodium alginate to soften water is more suitable for cultivation of H.pluvialis,which greatly improves the production of H.pluvialis and the accumulation of astaxanthin,providing a theoretical basis for the large-scale cultivation of H.pluvialis in Chenghai area.Astaxanthin derived from H.pluvialis is considered to be the safest natural astaxanthin.It has been widely used in medical,food and health products,feed and other industries with good results.However,due to the particularity of the pregnant population,the safety and caution of medication,there is no report on the safety of astaxanthin in the pregnant population.Our study is the first to explore the metabolism,distribution and safety evaluation of astaxanthin from H.pluvialis in pregnant mice.In the acute toxicity test,the experiment was divided into blank control group,astaxanthin low-dose group and astaxanthin high-dose group.Astaxanthin was administered intragastrically to pregnant mice according to the dose.After 2 weeks of observation,it was found that astaxanthin had no acute toxicity to pregnant mice,and the oral astaxanthin LD₅₀ was greater than20g/kg·bw.In the bone marrow micronucleus test,the experiment was divided into blank control group,astaxanthin low-dose group,astaxanthin medium-dose group,astaxanthin high-dose group and positive control group.Astaxanthin was administered intragastrically to mice 1 week after pregnancy and detected 48 hours later.The results showed that10g/kg·bw astaxanthin had no damage to chromosome and mitotic apparatus in pregnant mice.When 500mg/kg·bw of astaxanthin was given to mice,the blood astaxanthin content reached the peak value of 55.7μg/L at 8h,and the metabolism of astaxanthin in blood of mice was completed 48h later.In short-term repeated dose experiments,astaxanthin was administered to mice at 100,250 and 500mg/kg·bw.It was found that astaxanthin did not cause changes in body weight,organ weight,blood routine and biochemical indexes of pregnant mice during the whole pregnancy.Astaxanthin accumulated most in the liver and least in the eyes throughout the pregnancy of the mice.All the above results indicate that astaxanthin from H.pluvialis has no adverse effects on pregnant mice,and has the potential to be used in pregnant women.In recent years,with the improvement of people’s living standards,the incidence of diseases among pregnant women has also been high.The powerful antioxidant and anti-inflammatory effects of astaxanthin have been fully verified in the prevention and treatment of diseases in normal people.Can astaxanthin also significantly improve pregnancy diseases such as gestational diabetes（GDM）?We used streptozotocin（STZ）to establish a GDM model in pregnant mice to explore the preventive effect of astaxanthin on GDM and its possible mechanism.The experiment was divided into blank control group,streptozotocin（STZ）treatment group,STZ+astaxanthin low-dose group and STZ+astaxanthin high-dose group.The effects of astaxanthin on fasting blood glucose,insulin,insulin resistance in liver and insulin signaling pathway,glucose transport in liver and placenta,and antioxidant inflammation in pregnant rats were investigated.The results showed that astaxanthin reduced fasting blood glucose and insulin,improved the development of glucose intolerance and insulin resistance in GDM mice,and restored insulin-mediated glucose transport through NOX4-ROS-iκB mediated IRS-1/PI3K/Akt pathway.Meanwhile,astaxanthin reduced oxidative stress and inflammation level in the placenta and liver,and improved glucose transport mediated by insulin-independent pathways.This study confirmed that astaxanthin can effectively improve the occurrence and development of GDM,and provides a theoretical basis for the application of astaxanthin in pregnancy diseases.GDM is thought to be related to the oxidative damage of placental vascular endothelial cells.The role of astaxanthin in endothelial cell antioxidation is unclear.In order to explore the potential effects of astaxanthin on endothelial cells,we used human umbilical vein endothelial cells（HUVEC）as the research object to explore the antioxidant mechanism of astaxanthin in HUVEC.The experiment was divided into control group,astaxanthin low-dose,medium-dose and high-dose groups.The effective dose and action time of astaxanthin in HUVEC cells,the effects on cell survival rate,reactive oxygen species（ROS）,antioxidant enzymes,MAPK,Nrf2/ARE/HO-1 signaling pathway were mainly explored.The results showed that the optimal action time of astaxanthin in HUVEC was more than 18h,and the effective concentration was less than 0.05%.0.1,1 and 10μM astaxanthin increased intracellular ROS by 9.35%,14.8%and 18.06%,respectively.This ROS production did not cause cell death,but improved the cell survival rate and up-regulated the activity of II enzymes HO-1 and GSH-Px.In addition,after astaxanthin treatment,we observed phosphorylation of ERK protein,transfer of Nrf2 protein into the nucleus,and increase of ARE luciferase activity.After Nrf2 was interfered with by siRNA,HO-1mRNA expression was inhibited by 60%,indicating that astaxanthin activated the Nrf2/ARE signaling pathway.Our results indicate that astaxanthin activates the Nrf2/HO-1antioxidant signaling pathway through the production of trace ROS.In conclusion,sodium alginate softened water was used in this study to improve the yield and astaxanthin accumulation of H.pluvialis,which provided a new idea for the large-scale cultivation of H.pluvialis in Chenghai area.At the same time,the safety and effectiveness of astaxanthin in pregnant mice and GDM were clarified,which provided a theoretical basis for the treatment of diseases during pregnancy,and also provided a new idea for the development of astaxanthin products.