Analytical MethodsDetermination of carotenoids in Dunaliella salina cultivated in Taiwan and antioxidant capacity of the algal carotenoid extract
Introduction
Dunaliella salina is a type of unicellular and halophilic green biflagellate microalga without a rigid cell wall structure (Ben-Amotz and Avron, 1992, Denery et al., 2004, García-González et al., 2005, Raja et al., 2007). Because D. salina contains abundant β-carotene, the algae has been used as a food coloring agent, a pro-vitamin A supplement for food and animal feed, an additive to food and cosmetics, a health food product (antioxidant claim) and so on (Edge et al., 1997, Johnson and Schroeder, 1995).
Carotenoids are recognized as efficient antioxidants against oxidative damage (Jimenéz, & Pick, 1993). They could quench singlet oxygen (1O2), resulting in the suppression of lipid peroxidation (Burton and Ingold, 1984, Foote and Denny, 1968). Ben-Amotz (1999) indicated that humans could lower incidence of certain cancers, coronary heart disease and other degenerative diseases through eating carotenoid-rich vegetables and fruits (Ben-Amotz, 1999, Gester, 1993, Ziegler, 1989). For the determination of carotenoids, the reversed-phase high performance liquid chromatography (RP-HPLC) has been used routinely because of its excellent separation efficiency (Chen et al., 2004, Inbaraj et al., 2006, Liu et al., 2004, Tai and Chen, 2000). There are, however, no thorough reports on the composition and content of carotenoids in D. salina cultivated in Taiwan.
In the present study, we developed an isocratic RP-HPLC method to determine carotenoids including their isomers in D. salina; the amounts of these compounds in the algae were also quantified. The antioxidant activities of the algal carotenoid extract for Trolox equivalent antioxidant capacity (TEAC) assay, reducing power and scavenging ability on 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) radicals were also investigated in this work.
Section snippets
Materials
Spray dried powder of D. salina (Dunal) Teod. cultivated in Taiwan was obtained from Gong Bih Enterprise Co., Ltd. (Wunlin, Taiwan).
Chemicals and standards
All-trans forms of α-carotene, β-carotene, lutein and zeaxanthin standards were purchased from Sigma Co. (St. Louis, MO, USA). Solvents used for the extraction and determination of carotenoids, such as acetonitrile (ACN), methanol (MeOH), methylene chloride (CH2Cl2), ethanol (EtOH), acetone and n-hexane were purchased from Merck Co. (Darmstadt, Germany). Deionized
HPLC analysis of carotenoids in D. salina
Fig. 1 is the HPLC chromatogram of carotenoid extract from D. salina. Seven carotenoids (the spectral characteristics and Q-ratios of the seven resolved peaks corresponded to carotenoids) in the extract could be separated simultaneously within 30 min. Table 1 shows the assignment data for all-trans and cis forms of carotenoids in D. salina. Peaks 1, 2, 4 and 6 were certainly assigned as all-trans forms of lutein, zeaxanthin, α-carotene and β-carotene, respectively based on the criteria expounded
Conclusion
In this investigation, seven carotenoids were determined in D. salina cultivated in Taiwan using a HPLC method with isocratic solvent system. The algal carotenoid extract had remarkably higher antioxidant activity than all-trans forms of α-carotene, β-carotene, lutein and zeaxanthin. The cis forms of carotenoids in the extract, especially 9- or 9′-cis-β-carotene, might play crucial roles in the antioxidant activity. Therefore, our work provides the necessary information to exploit D. salina as
Acknowledgment
This work was supported by Chun Shan Medical University, Taichung, Taiwan (Project No. CSMU-82-B-013 and CSMU 94-OM-A-163).
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