Polymethoxylated flavones

Normal phases (unmodified silica gel) are rarely employed, except for the occasional separation of weakly polar flavonoid aglycones, polymethoxylated flavones, flavanones, or isoflavones. The polymethoxylated flavones present in citrus fruits can, for example, be separated on silica gel columns. The big drawback is that solvent gradients cannot normally be run with normal phases. 

Berahia, T. et al.. Mass spectrometry of polymethoxylated flavones, J. Agric. Food Ghent., 42, 1697, 1994. 

Happi, E.N. and Mpondo, T.N., Two polymethoxylated flavones from Distemonanthus bentha-mianus, J. Nat. Prod., 57, 291, 1994. 

Takanaga H, Ohnishi A, Yamada S, et al. Polymethoxylated flavones in orange juice are inhibitors of P-glycoprotein but not cytochrome P450 3A4. J Pharmacol Exp Ther 2000 293(l) 230-236. 

Table 7 Gradient HPLC for the Simultaneous Analysis of Flavanone Glycosides and Polymethoxylated Flavones...

Detection UV 280 nm for flavanone glycosides UV 330 nm for polymethoxylated flavones Injection volume 20 /A... 

Dilute citrus juice (25 ml) with dimethylformamide (20 ml), steam-bath for 10 min at 90°C. Cooling, adjust the volume to 50 ml with water, centrifuge (10 min at 2500 g), and then filter (Acrodisc, 5 and 0.45 /tm) the clarified juice. For standards, prepare standards (narirutin, naringin, neohesperidin, didmin, and porcirin) with 30% DMF in water (10-80 ppm). Prepare hesperidin in 70% DMF in water (200 ppm). Prepare polymethoxylated flavones in methanol (25 ppm). 

Fig. 13 HPLC separation of flavanone glycosides and polymethoxylated flavones (PMFs) in orange juice spiked with didymin and PMFs.

Polymethoxylated flavones are almost exclusively found in citrus. The structure of some polymethoxylated flavones are shown in Fig. 14. The polymethoxylated flavones are an interesting group of bioactive compounds that are concentrated on the fruit surfaces (peel) and are pres-... 

Polymethoxylated flavones are also characteristic of orange peel oils, and PMF determination has been used to ascertain the geographical origin of industrial peel oils (110). A normal-phase HPLC method for the determination of flavones in orange and mandarin oils was developed by Gaydou and co-workers (111). Using the normal-phase mode with a Licrosorb Si60... 

EM Gaydou, T Berahia, JC Wallet, JP Bianchini. Gas chromatography of some polymethoxylated flavones and their determination in orange peel oils. J Chromatogr 549 440-445, 1991. 

EM Gaydou, JP Bianchini, RP Randriamiharisoa. Orange and mandarin peel oils differentiation using polymethoxylated flavone composition. J Agric Food Chem 35 525-529, 1987. 

M Hadj-Mahammed, BY Meklati, Qualitative determination of polymethoxylated flavones in Valencia orange peel oil and juice by LC-UV/Vis and LC-MS techniques. Lebensm Wiss Technol 20 111-114, 1987. 

Substantial quantities of luteolin-7-O-glucuronide, luteolin-7-O-glucoside, and luteolin-7-O-rutinoside occur in Red Oak Leaf and Lollo Rosso, two red-leaved varieties of lettuce (Lactuca sativa) [Llorach et al., 2008], Polymethoxylated flavones such as nobiletin, scutellarein, sinensetin, and tangeretin (Fig. 1.8) are found exclusively in citrus species [Crozier et al., 2006c], while diosmetin-7-O-glucuronide has been isolated from the fruits of a Chinese herb, Luffa cylindrical. 

Figure 1.8 Polymethoxylated flavones found in citrus species.

These compounds are differentiated primarily by the oxidation state of the central three carbon atom unit. Thus, as shown in Figure 1, some compounds are classified as flavones, flava-nones, flavonols, anthocyanins, etc. (The A and B rings have been left off for clarity). A secondary means of differentiating flavonoids is by the position and numbers of attached hydroxy, methoxy or sugar units. In citrus, flavonoids usually occur as glycosides, although the polymethoxylated flavones are a notable exception. 

Compositions of volatiles in different orange oils are shown in Tables 4 and 5. It is evident that most of the constituents belong to the terpene family and may be arranged into two groups, terpene hydrocarbons (terpenes and sesquiterpenes) and oxygenated terpene products (21). Aside from the volatile components, there are small amounts (2-15%) of nonvolatile residues in citrus peel oils that possess antioxidative property these include coumarins, psoralens, and polymethoxylated flavones (30-34). 

Citrus peel oils may also be used for their antioxidative, antitumor, and radicalscavenging activities. The radical-scavenging ability of citrus peel oil may help prevent free radical-induced and various chronic diseases (48, 55, 56). Monoterpenes from volatile components and polymethoxylated flavones from nonvolatile residues have been reported to be effective inhibitors of tumor cell growth, implicating that citrus peel oils may be good cancer preventive food additives (57-59). Furthermore, citrus peel oils are useful to alleviate pain from burnt skin (60). Demonstrating anxiolytic and sedative effect, they could also be used in primary medical care against insomnia, anxiety, and epilepsy (61). 

Several kinds of flavonoids are efficiently separated and analyzed using packed or capillary column supercritical fluid chromatography. The composition of mobile phase, stationary phase, temperature, and pressure aU affect the resolution. This article mainly focuses on the separation of polymethoxylated flavones, polyhydroxyl flavonoids, and flavonol isomers. 

Traditionally, flavonoids have been separated and analyzed by HPLC and gas chromatography (GC). However, recent developments of SFC may permit a more accurate and complete analysis of plant phenolic compounds. Supercritical fluid chromatography brings together the advantages of both HPLC and GC techniques because it may be readily employed in the analysis of nonvolatile and thermolabile compounds and provides facile coupling to detector technologies such as mass spectrometry and Fourier transform infrared (FT-IR) spectroscopy. In recent years, SFC has been used to separate flavonoid compounds, most of which are polymethoxylated flavones and polyhydroxylflavonoids. 

Morin et al. successfully separated polymethoxylated flavones (PMFs) by packed-column SFC, illustrating that the SFC procedure is considerably faster than HPLC, with good resolution and adequate accuracy for the quantitative analysis of the PMFs. The chromatographic system... 

Morin, P. Gallois, A. Richard, H. Gaydou, E. Fast separation of polymethoxylated flavones by carbon dioxide supercritical fluid chromatography. J. Chromatogr. 1991, 586, 171-176. 

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