Mobile phase additives for enhancing the chromatographic performance of astaxanthin on nonendcapped polymeric C30‐bonded stationary phases

Astaxanthin shows peak deformation and reduced peak area response when eluted with methanol and methyl tert‐butyl ether on nonendcapped polymeric C₃₀‐bonded HPLC phases. The present study tested different column manufacturers, column batches, and ten mobile phase additives including acids, bases, buffers, complexing and antioxidant agents for improvement of peak shape and peak area response. Concerning chromatographic benefits and feasibility, ammonium acetate was found to be the best additive followed by triethylamine for all columns tested. Variation of the mobile phase pH equivalent and the column temperature showed no synergistic effects on peak shape and peak area response. Results indicate that peak tailing and variation of peak area response are due to different on‐column effects. Possible mechanisms of the observed phenomenon will be discussed.     

超临界CO2萃取-反相高效液相色谱测定海南对虾壳中虾青素的含量

采用超临界CO2萃取技术,研究了从海南对虾壳中提取虾青素的工艺,同时采用正交实验方法研究了其最优化萃取条件,结果表明:萃取压力为35 MPa,萃取温度为60℃,夹带剂为100g样品用15 mL二氯甲烷,萃取时间为2 h.最后用高效液相色谱法对萃取产物中的虾青素进行了测定,色谱柱:waters novapakC18 3....     

Optimization of acidic extraction of astaxanthin from Phaffia rhodozyma

Optimization of a process for extracting astaxanthin from Phaffia rhodozyma by acidic method was investigated, regarding several extraction factors such as acids, organic solvents, temperature and time. Fractional factorial design, central composite design and response surface methodology were used to derive a statistically optimal model, which corresponded to the following optimal condition： concentration of lactic acid at 5.55 mol/L, ratio of ethanol to yeast dry weight at 20.25 ml/g, temperature for cell-disruption at 30 ℃, and extraction time for 3 min. Under this condition, astaxanthin and the total carotenoids could be extracted in amounts of 1294.7 μg/g and 1516.0 μg/g, respectively. This acidic method has advantages such as high extraction efficiency, low chemical toxicity and no special requirement of instruments. Therefore, it might be a more feasible and practical method for industrial practice.     

Purification of Astaxanthin from Laminaria japonica by Ionic Liquid-based Monolithic Cartridge

An effective and accurate method was developed for the extraction of astaxanthin from Laminaria japonica with subsequent separation by ionic liquid-based monolithic cartridge. The optimized extraction conditions including extraction solvent(ethanol), extraction time(90 min) and ultrasonic power(75 W) were obtained by systematical investigation. Chromatographic analysis was performed on a C₁₈ column with ultraviolet(UV) detection at 476 nm, and a solution consisting of methanol/acetonitrile/H₂O/dichloromethane(83:6:6:5, volume ratio) was used as the mobile phase at a flow rate of 0.7 mL/min. After ionic liquid-based monolithic solid phase extraction, 17.82 μg/g astaxanthin was obtained from Laminaria japonica. This ionic liquid-based monolithic cartridge exhibits high affinity and selectivity for astaxanthin, and it can be potentially used as the stationary phase of high performance liquid chromatography(HPLC).     

雨生红球藻的培养及虾青素的提取与检测

以雨生红球藻为原材料,通过对光照条件、培养液pH和温度等条件的优化确定雨生红球藻在改良后的BBM培养基中的适宜生长条件;采用研磨结合超声波破碎的方法对雨生红球藻进行破壁,利用乙酸乙酯、丙酮和70%乙醇分别提取雨生红球藻中的虾青素,并通过紫外吸收光谱、红外吸收光谱以及高效液相色谱对虾青素的含量进行检测分析,确定虾青素的最佳提取方法。结果表明:在pH=8.0的改良BBM培养基中,光照度为2 000~2 500 lx时连续24 h光照条件下雨生红球藻能够快速生长和繁殖,采用研磨法破壁后利用乙酸乙酯提取虾青素的提取率可达91.41%。本实验优化了雨生红球藻的培养条件,确定了虾青素提取的有效方法,为雨生红球藻的扩大生产及深入研究奠定了基础。     

Determination of astaxanthin in feeds using high performance liquid chromatography and an efficient extraction method

A high performance liquid chromatography method using an efficient extraction method was developed for the determination of astaxanthin in eight kinds of animal feed. The chromatographic separation was achieved using a C₁₈ reversed-phase column, using methanol and acetonitrile as the mobile phases with a flow rate of 1 mL/min. The feeds containing astaxanthin were first treated with maxatase, to cause enzymatic hydrolysis, and then extracted with dichloromethane. The optimized method produced recoveries of between 82.4% and 100% for all eight kinds of feed, and the coefficients of variation were lower than 4.28%. The limit of detection, defined as the concentration that gave a signal-to-noise ratio of 3, for astaxanthin was estimated to be 0.1 µg/g. The limit of quantification, defined as the lowest spiked concentration that gave an appropriate level of precision and accuracy, was 0.3 µg/g. Finally, the method developed was used to determine astaxanthin in real, commercially sourced, feed samples. The method met the requirements for the determination of astaxanthin in feed, providing satisfactory recoveries of 70–110%.     

Optimization and Application of Liquid Chromatography Determination of Dispersive Liquid-liquid Microextraction Purified Astaxanthin in Shrimp Waste

A new molecularly imprinted solid-phase extraction(MISPE) monolithic cartridge was synthesized, and MISPE-DLLME(DLLME=dispersive liquid-liquid microextraction) was developed for purification of astaxanthin in shrimp waste. The eluent(methanol) from MISPE was used as the dispersive solvent in subsequent DLLME for further purifying and enriching the analyte prior to high-performance liquid chromatography(HPLC) analysis. The mobile phase was methanol-acetonitrile-water-dichloromethane(85:5:5:5, volume ratio), flow rate was 0.7 mL/min and UV wavelength was 476 nm. Under optimal conditions, good linearity was obtained in a range of 0.2―200.0 μg/mL(r²=0.9998) with a limit of detection(LOD) of 0.08 μg/mL, and the extraction recoveries at three spiked levels ranged from 88.3%―92.5% with a relative standard deviation(RSD) less than 4.3%. Moreover, the mean contents of astaxanthin in the three batches of shrimp waste were 95.9, 85.4 and 77.2 μg/g, respectively. This method combining the advantages of MISPE and DLLME results in high selectivity and low cost, which was applied to determining the astaxanthin level in shrimp waste samples.     

虾青素抑制H2O2诱导的HeLa细胞凋亡

为了阐明虾青素的抗氧化作用与细胞凋亡的关系,探究虾青素预处理对H_2O_2诱导HeLa细胞氧化应激的影响.通过CCK-8、活性氧探针染色、流式细胞术、蛋白质免疫印迹、实时荧光定量pcr等,分别检测细胞存活率和活性氧的积累、细胞凋亡、蛋白含量、基因相对表达量改变.结果表明虾青素预处理组细胞活力较对照组提高了29.54%以上且其可以将H_2O_2诱导的活性氧降低至对照水平,同时提高Nrf2蛋白表达量3倍之多,过氧化氢酶基因相对表达量1.5倍.说明虾青素可以有效缓解H_2O_2诱导的HeLa细胞氧化应激,从而抑制细胞凋亡.     

化学因子对雨生红球藻诱变株虾青素积累的调控

在温度为(20±1)℃,光照为40μEm-2s-1条件下对雨生红球藻经紫外诱变所得的藻株1号、藻株2号及对照株进行比较培养,结果表明:诱变株1号生长速率最快.进一步对诱变株1号进行缺氮、缺磷、缺铁、添加醋酸对其虾青素累积速率影响的试验以及氮、磷、铁的三因子三水平正交试验.其中氮试验中,分别设置0,1,2,3,4,5 mg/L 6个梯度进行实验.磷试验中分别设置0,0.5,2,4,8,10 mg/L 6个梯度进行实验.铁试验中,分别设置0,0.1,0.2,0.4,0.5,1 mg/L 6个梯度进行实验.添加醋酸试验分别设置0,0.5,1,1.5,2,2.5 mg/L 6个梯度进行实验.结果表明,在氮、磷、铁完全缺乏的情况下和醋酸浓度为2 mg/L下,红球藻的虾青素累积速率最高.三因子三水平正交试验里表明氮、磷、铁3个因子对虾青素的累积都有极显著的促进作用,其中氮磷、氮铁之间的交互作用显著.氮胁迫能诱导雨生红球藻从绿色游动细胞转变成红色不动细胞,培养基中磷胁迫和醋酸钠刺激能大大加快这一转化的速率.     

Optimization of ultrasonic-assisted extraction of astaxanthin from green tiger (Penaeus semisulcatus) shrimp shell

This study was aimed at optimizing the astaxanthin extraction efficiency from shrimp shell (green tiger, Penaeus semisulcatus). Astaxanthin was extracted using selected nonpolar/polar solvents (petroleum ether, n-hexane, ethanol, acetone) individually and in ternary mixtures of petroleum ether, acetone, and water in ratios of 15:50:35, 50:45:5, and 15:75:10 for different times (2,4 and 6 h). The results showed that solvents with higher polarity were more suitable for the extraction of astaxanthin, and increasing the extraction time from 2 to 6 h improved the extraction yield. The conditions of extraction of astaxanthin with the desirable solvent were then optimized with the ultrasonic method using the Box-Behnken design [variables included: extraction temperature (25 to 45 °C), extraction time (5 to 15 min), and ultrasound amplitude (20 to 100%)]. Optimal extraction conditions were determined as the ultrasonic amplitude of 23.6%, extraction time of 13.9 min, and extraction temperature of 26.3 °C. Under this optimum condition, the amount of astaxanthin, ferric reducing antioxidant power, and free radical scavenging capacity of the extract were obtained as 51.5%, 1705 μmol of Fe²⁺/g, and 73.9%, respectively. Extraction and analysis of the extract at the optimum point were used to validate the results.