**4.1 Spectral characteristics of flavonoids**

UV spectroscopic analysis of flavonoids identified two major absorption bands: Band I (320–385 nm) representing the absorption of the B ring, and Band II (250–285 nm) representing the absorption of the A ring. A shift in absorption can occur due to functional groups attached to flavonoid skeletons, such as 367 nm for kaempferol (3,5,7,4′-hydroxyl groups) and 371 nm for quercetin (3,5,7,3′,4′-hydroxyl

#### *Chemistry and Role of Flavonoids in Agriculture: A Recent Update DOI: http://dx.doi.org/10.5772/intechopen.106571*

groups) and 374 nm for myricetin (3,5,7,3′,4′,5′-hydroxyl groups) [53]. An absence of a 3-hydroxyl group distinguishes flavones from flavanols. According to their UV spectral properties, flavanones have a saturated heterocyclic C ring with no conjugation between the A and B rings [54]. Flavanones show only a shoulder for Band I at 326 and 327 nm and a very significant Band II absorption maximum between 270 and 295 nm, namely 288 nm for naringenin and 285 nm for taxifolin. In compounds with a monosubstituted B ring, Band II shows one peak (270 nm), but when a di-, tri-, or o-substituted B ring is present, it shows two peaks or one peak (258 nm) with a shoulder (272 nm). The color of anthocyanins varies with the quantity and position of the hydroxyl groups because they exhibit discrete Band I peaks in the 450–560 nm area due to the hydroxyl cinnamoyl system of the B ring and Band II peaks in the 240–280 nm region due to the benzoyl system of the A ring [55].

Nuclear magnetic resonance (NMR) spectroscopy has proven essential in the structural elucidation of natural products; it is one of the most effective methods available to natural product chemists [10].

In <sup>1</sup> H NMR investigations, the chemical shifts (δ) and the coupling constants (*J*), also known as and spin–spin couplings, are a good indicator. By comparing the recorded chemical shifts with the gathered data, this parameter provides important information on the relative number and kind of hydrogens. The number and anomeric configuration of the glycoside moieties connected to the aglycone, as well as the aglycone and acyl type groups associated to it, may all be determined using this [10]. The molecular architecture of flavone (Itoside N) can be learned a lot from the analysis of its 1H NMR spectrum data. The presence of an aromatic proton at C-3 in ring-C is shown by the one-proton singlet that appears at δ 6.86. The presence of two aromatic protons at C-6 and C-8, respectively, in ring-A is indicated by the signals that occurred as doublets (d) at δ 6.46 (1H, d, *J* = 2.0 Hz) and 6.80 (1H, d, *J* = 2.0 Hz). Four aromatic protons of the B-ring in the flavone skeleton may be the cause of the doublet (d) signals that emerged at δ 7.97 (2H, dd, *J* = 8.0 Hz) for two protons and δ 6.97 (2H, dd, *J* = 8.0 Hz) for another pair of protons. Ring B is definitely paradisubstituted, according to the chemical shifts and coupling constant values for its four protons. Moreover, the 1 H NMR spectrum of Itoside N (**Table 1**) indicates that a partial structure similar in structure to p,p′-dihydroxy-μ-truxinic acid in Itoside N is formed by two p-dihydroxy benzenoid groups that combination with a cyclobutane [δ 42.9 (C-2′′′), 43.5 (C-3′′′), 45.5 (C-2′′′′), 42.9 (C-3′′′′)] moiety [9, 10].

When combined with 1 H NMR data, 13C NMR data can be used to determine the types of groups that are present in molecules. It should be noted, however, that 13C NMR is not as responsive as 1 H NMR because 13C is less abundant (1.1%) than 1 H (99.9%) [10]. The C-2/C-II-2 and C-3/C-II-3 sp2-hybridized carbons can be found, respectively, at δC 152.5–165.5 and 103–132.1 in 13C-NMR spectra. According to 13C NMR data of flavonoid compounds, C-4/C-II-4 appear between δC 176.2 and δC 182.9 when C-2-C-3 is unsaturated (sp2), but when C-2/C-3 is sp3 hybridized, a down-field shift of C-4/C-I-4 is typically observed between δC 196.2 to δC 197.9. The typical range of δC 159.6–164.7 includes the aromatic C-5, C-6, C-7, C-8, C-9/C-4a, and C-10/C-8a. Generally CMR of glucopyranosyl moiety appeared in the range of δC 60.6–102.11; rhamnopyranosyl (Rha) also appeared in the range of δC 17.23–101.0; and glucuronopyranoside showed the value in δC 98.4–171.6. The value of glucuronopyranoside is nearly identical to that of the glucopyranosyl moiety, however the carboxylic group is what gives the compound its high value (δC 171.6). The flavone, Itoside N is found to appear around a range δC of 42.9–172.2 for the 4′′/6′′-p-hydroxy-μ-truxinyl group [10].

**9**
