| Marigold (Tagetes erecta L.) is a common ornamental plant which bears yellow to orange flowers. Its flower petals are an excellent and one of the most important sources of lutein. The bioactive component of lutein is used as feed additive, nutritional supplement and even medicine. Reported potential benefits of lutein include the prevention of eye diseases (such as Age-Related Macular Degeneration, AMD), enhancement of immune function, and protection against oxidation induced cell damage. In order to comprehensively explore the characters of lutein in marigold, the fingerprint-chromatogram, stability, hydrolysis technology, quantification methods of lutein, and food safety assessment about pesticide and metal residues were studied. The main conclusions obtained were listed as follows:(1) Five carotenoids, six lutein diesters and three lutein monoesters, were purified by thin liquid chromatography (TLC) and identified by comparing the spectrum data and retention time with the reported data. The fingerprint-chromatogram of marigold hexane extracts was developed by HPLC. The fingerprint-chromatograms of petal, powder, granule, feed additive, milk additive and capsule samples were analyzed, and it was the six lutein diesters that could be regarded as the characteristic component of marigold products. The free lutein, cis-lutein and lutein monoester was seldom found in fresh petal and marigold powder, however cis-lutein and lutein monoester existed in feed and food additive.(2) The stability of lutein at different pH, temperature, oxygen content conditions and in different organic solvents was studied. The results showed that lutein was stable in weak acid and weak base environment. When the temperature was80℃or lower, lutein was not sensitive to heat. The storage time of lutein was prolonged with antioxidant and nitrogen protection. The degradation rates of lutein in different organic solvents fitted the first order reaction model. In dichloromethane, two cis-luteins were formed, and the total amount fitted the first order reaction model, whereas, the amount of single cis-lutein did not fit any kinetic equations in ethanol. In ethyl acetate, the degradation of9(9’)-lutein+13(13’)-lutein fitted the reversible second-order model, but the trend was different with that in dichloromethane. The results showed that the trans-cis isomerization in different solvents excitated by light was diversiform.(3) The heat reflux hydrolysis of lutein esters was studied. The method was optimized with four factors such as temperature, time, alkali concentration and organic solvent. Lutein esters were also treated with cold saponification and ultrasonic technologies. The results showed that both technologies were not able to hydrolyze lutein esters fast and completely. Hydrolysis of lutein esters with microwave was studied as well, and the degradation and isomerisation rate was estimated. The results showed that lutein esters were hydrolyzed in a shorter time and with a smaller isomerization rate, which means microwave will be a good choice of hydrolyzing lutein esters in the future.(4) The quantification methods of lutein were researched. The AOAC method was optimized, in which the toxic solvent of benzene was replaced by2-propanol. The total lutein amount was determined by Lambert-Beer law. The absorbence values of lutein and lutein esters were assessed. Data showed that the regression coefficients between lutein and lutein esters were the same, and the absorbence value of lutein ester was8%higher than that of lutein. A standard method for lutein determination (NY/T2008-2011) was built by HPLC. The repeatability of the method was performed intraday, interday and by two analysts. The accuracy of the method was evaluated by a recovery study. The reproducibility was performed at different laboratories. The data was analyzed according to GB/T6379.2-2004Chinese standard. Results indicated that the method was simple, highly sensitive and suitable for routine analysis. And for the first time, lutein and zeaxanthin were separated by ultra-performance liquid chromatography (UPLC), with a reduced analyzing time of3min. Resolution obtained by UPLC was twice higher than HPLC reported in literature. Sensitivity was expected to be higher by UPLC than that by HPLC. For lutein, the limits of detection in two methods were the same, whereas the limit of detection for zeaxanthin by UPLC was twofold lower than that by HPLC. The concentrations of lutein and zeaxanthin in twenty marigold samples were assessed by UPLC method and HPLC method, a Mest of paired measurements at a significance level of α=0.05showed that there was no significant difference between the two methods.(5) The food safety of marigold and its products was assessed. A device to distill and absorb pesticide was set up, which could determine the total amount of dithiocarbamatre in marigold. The derivative reaction was carried out by equipment consists of two pumps and a temperature control unit, which could analyze7carbamates in marigold. A new derivative mechanism of N-methylimidazole (MI) reacting with trifluoroacetic anhydride (TFAA), then reacting with the hydroxyl group of abamerctin to make fluorescence was supposed. It implied that flu-OH was not competely derived from flu-TFA hydrolysis. Some pesticides such as methamidophos were found in marigold by these optimized methods. So it was imperative to constitute rules about Good Agricultural Practice (GAP). The metal elements of As, Hg, Pb and Cd were analyzed by microwave digestion followed by atomic fluorescence spectrophotometer or atomic absorption spectrophotometer. The results showed that there might be risk of As and Cd exceeding certain limits in marigold.In conclusion, the chemical property, degradation kinetics and quantification methods of lutein, microwave hydrolysis of lutein esters, and food safety assessment about pesticide and metal in marigold products were studied in this paper. A Standard numbered NY/T2008-2011, in which lutein in marigold and products was determined by HPLC, was issued by the minister of agriculture, China for the first time. The planting of marigold can be guided and its competitiveness in the international market can be intensified by this standard. |
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