Analytical, Nutritional and Clinical MethodsSeparation and identification of the yellow carotenoids in Potamogeton crispus L.
Introduction
Carotenoids are important biological compounds that are present in all green tissues of higher plants. Carotenoids have many functions such as antioxidation, immunity-regulation, inhibiting proliferation of tumor cells etc. The applications of carotenoids in medicine and cosmetics have been well documented as is their utilization as food additives (colorants, antioxidants).
Potamogeton crispus L. (P. crispus) is a submersed herbaceous perennial plant, which could be found in freshwater lakes, ponds, paddy fields and rivers all over the China. It is an important primary producer in freshwater ecosystems, providing good fodder source for herbivorous fishes and poultry (Jian, Li, Wang, & Chen, 2003). We found that ducks fed with P. crispus could lay natural red-yolk eggs with good quality. It is known that the color of egg yolk is closely related with the type and concentration of carotenoids in feed. Chemical analytical study and feeding trials proved that carotenoids in the red-yolk eggs mainly derived from P. crispus (Wang et al., 2003). P. crispus is a new resource of carotenoids, while very few reports were about the identification and classification of carotenoids in P. crispus. The red carotenoids in P. crispus were separated and identified by thin layer chromatography (TLC), HPLC-MS/MS, Raman micro-spectroscopy, FT-IR sepectroscopy and high resolution EI-MS in our lab. The results showed that the red carotenoids were rhodoxanthin and its cis/trans isomers (Liu et al., 2004, Wang et al., 2004).
Nowadays methods for carotenoids isolation and analysis include thin-layer chromatography (TLC), open column chromatography (OCC) and high-performance liquid chromatography (HPLC). For identification and structure elucidation, visible spectrophotometry, NMR and mass spectrometry are the most recommended (Azevedo-Meleiro and Rodriguez-Amaya, 2004, de Sá and Rodriguez-Amaya, 2003, Hodisan et al., 1997, Lacker et al., 1999, Oliver and Palou, 2000).
Section snippets
Chemicals
β-carotene standard and diatomaceous earth are purchased from Sigma Company, USA. Most of solvents and chemicals were of analytical grade (Magnesium oxide, sodium sulfate anhydrous, anhydrous sodium carbonate, methanol, petroleum ether, acetone, anhydrous ethanol, acetone, chloroform, n-hexane, carbon bisulfide, BHT). Methanol was of HPLC grade purchased from Fisher Chemicals Company, USA. Silica gel G60 for thin-layer chromatography was obtained from Qingdao Haiyang chemical company, China.
Sample extraction and saponification
Purity of the isolated fractions
The HPLC chromatograms of the isolated carotenoids fractions showed that each fraction has a single peak. Photodiode array measurements of spectral properties for the individual peaks were determined at the upslope, apex and downslope. The matching of the three spectra indicated the degree of peak purity. The results showed the purity of fractions was highly satisfactory.
TLC of carotenoids from P. crispus
The thin layer chromatogram of TCPC and B1, B2 and B3 obtained from TCPC by column chromatography showed that five fractions
Conclusions
P. crispus is a new resource of carotenoids. In this study, four major yellow carotenoid pigments in P. crispus were separated by a MgO column and thin-layer chromatography. Theose carotenoids were identified as neoxanthin, violaxanthin, lutein and β-carotene based on visible spectra in different solvents compared with values reported in the literature, functional group tests and mass spectrum by LC-MS. The content which measured by visible absorption spectroscopy to be 231 μg/g (dry weight)
Acknowledgement
This work was supported by a grant from the Ph.D. Programs Foundation of Ministry of Education of China (No. 20020504008).
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