Flavonoids

UV spectra with absorption maxima at 240-280 nm and 300-380 nm together with characteristic mass spectra indicated flavonoid structures in the case of nine compounds of the Corylus extracts (Fig. 36). From the accurate molecular mass and formula given by ESI-TOF, and fragmentation patterns acquired by collision-induced dissociation (CID) in ESI-MS/MS analyses compared to authentic standards and to literature data (Ablajan et al. 2013, Ablajan et al. 2006, Cuyckens and Claeys 2004, Fabre et al. 2001) it was possible to characterise the structures of the flavonoids, although the applied MS/MS method was not suitable for the accurate identification of the molecules where no matching standards were available. Myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside and kaempferol were identified by comparing their chromatographic and spectrometric data to authentic standards, while in the case of the other six flavonoid

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compounds structural characterisation was carried out by comparison of the (-) ESI-MS/MS fragmentation to aglycone standards or to glycosides with the same aglycone but different sugar part, although it has to be noted that the unambiguous identification of the sugar moieties is not possible with the current MS/MS method.

Besides this fact, according to literature data (Ablajan et al. 2006) the fragmentation behavior of flavonoid-O-glycosides strongly correlates with the glycosylation position.

The cleavage of the glycosidic bond in deprotonated myricetin-, quercetin- and kaempferol-glycosides provide both radical aglycone ion ([Y0-H]^-•) and aglycone ion (Y0

-) products. From the relative abundance of the aglycone ion it is possible to draw conclusions about the glycosilation position. Furthermore it has also been reported (Ablajan et al. 2013) that the type of the sugar moiety linked to the 3-O position has a certain effect on the formation and relative abundance on the [Y0-H]^-• ion. Thus following optimization of the collision energy (CE) during the negative ion ESI-MS/MS analyses conclusions about both the glycosilation site and type of the sugar moiety in the flavonol-glycosides could have been drawn.

Since negative ion ESI-MS/MS characterisation of flavonoid aglycones and glycosides is well reported (Ablajan et al. 2013, Ablajan et al. 2006, Cuyckens and Claeys 2004, Fabre et al. 2001), explanation of fragmentation behavior is not discussed here in full details.

Figure 36. The flavonoid compounds of the Corylus extracts.

*in the case of compound 28 the accurate position of the R3 group could not be identified

It has to be mentioned here that C. avellana leaves have already been reported to contain myricetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, a

quercetin-3-O-87

hexoside and kaempferol-3-O-rhamnoside (Amaral et al. 2010), while no previous literature data was found about the presence of these compounds neither in the other two Corylus species nor in the bark of C. avellana.

Myricetin-3-O-rhamnoside (21) was identified in the extracts by comparison of chromatographic and mass spectrometric behaviour to those of an authentic standard, and also by spiking the sample solutions with the standard in two different chromatographic methods (see sections 4.6.1. and 4.6.2.). The compound provided deprotonated molecular ion [M-H]^- at m/z 463.0871, aglycone fragments at m/z 316.0 ([Y0-H]^-•) and 316.9 (Y0

-) and other characteristic product ions at m/z 287 and 271. The ESI-TOF conjunction analysis and the molecular formula calculation correspond to the formula C21H20O12. The neutral loss of 146 amu confirmed the presence of a desoxyhexose sugar part. The product ion at m/z 316.0 ([Y0-H]^-•) was found to be relatively abundant in the (-) ESI-MS/MS spectra of both the compound and the reference standard. According to literature data (Ablajan et al. 2006), these observations strongly indicate the sugar moiety being located in 3-O position (in the case of flavonol-7-O-glycosides product ion [Y0-H]^-• is not intense). These observations were applied for characterisation of the other myricetin-glycoside (compound 22) in the extracts where no matching standard was available.

Quercetin-3-O-rhamnoside (23) was identified in the extracts by comparison of chromatographic and mass spectrometric behaviour to those of an authentic standard, and also by spiking the sample solutions with the standard in two different (see sections 4.6.1. and 4.6.2.). The compound provided deprotonated molecular ion [M-H]^- at m/z

88 spectrum). These observations were used for the characterisation of the quercetin-glycosides in the extracts where no matching standards were available.

Quercetin-3-O-hexoside (24) provided molecular ion [M-H]^- at m/z 463.0871, draw any appropriate conclusions about it.

Quercetin-3-O-glucuronide (25) exhibited deprotonated molecular ion [M-H]^- at m/z 477.0692, aglycone fragments at m/z 299.9 ([Y0-H]^-•) and 301.0 (Y0

-) and other characteristic product ions at m/z 271, 255, 179 and 151. The ESI-TOF conjunction analysis and the molecular formula calculation pointed to the formula C21H18O13. The neutral loss of 176 amu and the relatively high intensity of the product ion [Y0-H]^-•

suggested the presence of a glucuronide moiety in the 3-O position.

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Kaempferol (26) was identified in the extracts by comparison of chromatographic and mass spectrometric behaviour to those of an authentic standard, and also by spiking the sample solutions with the standard in two different chromatographic methods (see sections 4.6.1. and 4.6.2.). The molecular ion of kaempferol was detected at m/z 285.1700, the characteristic product ions at m/z 255 and 227.

Compounds 27, 28 and 29 exhibited the same aglycone fragment ion (m/z 285) and also the characteristic product ions at m/z 255 and 227 as kaempferol standard. In the MS spectrum of compound 27 the neutral loss of 146 amu pointed to a desoxyhexose moiety, in the case of compound 28 the neutral loss of 292 amu might indicate a di(desoxyhexose) sugar part, while in case of compound 29 the neutral loss of 176 amu suggested the presence of a glucuronide moiety. Therefore, compound 27 was tentatively identified as kaempferol-3-O-rhamnoside, compound 28 as kaempferol-di(desoxyhexoside) and compound 29 as kaempferol-3-O-glucuronide. In case of compound 27 the characterisation of the sugar part was based on literature data, kaempferol-3-O-rhamnoside had been previously reported from hazelnut leaves (Amaral et al. 2010).