Phytosterols occur in plants either as free or conjugated forms, the latter comprising steryl esters of fatty or phenolic acids and steryl glycosides. A mixture of esters, between hydroxycinnamic acids (CAD) and sterols based on the cholestane or the parent cycloartane skeletons, is collectively termed as "γ-oryzanol". Main CAD is ferulic acid; coumarate and caffeate esters of γ-oryzanol steroid moieties have been reported in the literature and we prepared them by syntheses. γ-oryzanol appears to lower the plasmatic level of low-density cholesterol and has anti-oxidant and free-radical scavenging properties. Several analytical methods have been proposed, most efforts have been devoted to RP-HPLC after pre-treatment of the samples. The analysis of γ oryzanol revealed some experimental drawbacks in the RP-HPLC methods, therefore we decided to explore the potential application of NP-HPLC in separating γ-oryzanol components. Our analysis was performed on cyanopropyl bonded column using a hexane/MTBE gradient system. A sample of standard γ-oryzanol was irradiated at UV light then subjected to NP-HPLC at semi-preparative scale; sample components were separated in two pairs of peaks. Each peak was collected and investigated by NMR spectroscopy and RP-LC-ESI-MS technique. The elucidation of 1H-NMR spectra of those fractions proved that our chromatographic method allows the separation of cis- from trans-ferulates (pairs eluting at shorter or longer tR respectively). The sensitive MS technique demonstrated that the peaks being eluted later in each pair contain steroid moieties which possess a double bond at the side chain whereas the peaks being eluted sooner in each pair contain compounds which are saturated at the side chain and, possibly, also at the polycyclic ring system. Natural γ-oryzanol contains saturated and unsaturated steroids which basically possess cholestane and cycloartane skeletons respectively. In conclusion, our method is suitable for quality assurance and determination of origin of γ-oryzanol and for its quantification as well.
Separation of g-oryzanol components and its synthetic p-coumarate and caffeate derivatives by NP-HPLC
D'Ambrosio, Michele
2012-01-01
Abstract
Phytosterols occur in plants either as free or conjugated forms, the latter comprising steryl esters of fatty or phenolic acids and steryl glycosides. A mixture of esters, between hydroxycinnamic acids (CAD) and sterols based on the cholestane or the parent cycloartane skeletons, is collectively termed as "γ-oryzanol". Main CAD is ferulic acid; coumarate and caffeate esters of γ-oryzanol steroid moieties have been reported in the literature and we prepared them by syntheses. γ-oryzanol appears to lower the plasmatic level of low-density cholesterol and has anti-oxidant and free-radical scavenging properties. Several analytical methods have been proposed, most efforts have been devoted to RP-HPLC after pre-treatment of the samples. The analysis of γ oryzanol revealed some experimental drawbacks in the RP-HPLC methods, therefore we decided to explore the potential application of NP-HPLC in separating γ-oryzanol components. Our analysis was performed on cyanopropyl bonded column using a hexane/MTBE gradient system. A sample of standard γ-oryzanol was irradiated at UV light then subjected to NP-HPLC at semi-preparative scale; sample components were separated in two pairs of peaks. Each peak was collected and investigated by NMR spectroscopy and RP-LC-ESI-MS technique. The elucidation of 1H-NMR spectra of those fractions proved that our chromatographic method allows the separation of cis- from trans-ferulates (pairs eluting at shorter or longer tR respectively). The sensitive MS technique demonstrated that the peaks being eluted later in each pair contain steroid moieties which possess a double bond at the side chain whereas the peaks being eluted sooner in each pair contain compounds which are saturated at the side chain and, possibly, also at the polycyclic ring system. Natural γ-oryzanol contains saturated and unsaturated steroids which basically possess cholestane and cycloartane skeletons respectively. In conclusion, our method is suitable for quality assurance and determination of origin of γ-oryzanol and for its quantification as well.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione
