**4. Structure characterization of flavonoids**

The structure characterization of flavonoids is related to the elucidation of their spectroscopic spectra obtained by techniques such as nuclear magnetic resonance (NMR), mass spectrometry (MS), spectrophotometric ultra-violet (UV) and infrared (IR). Physical properties of the flavonoids as melting point (mp), circular dichroism (CD), optical rotatory power ([α]D) are also useful for full characterization of the isolated flavonoid specially when its contains stereocenterfor CD and [α]D. Some chapters in this book would provide more details about the use of spectroscopic analysis to characterize flavonoids. Nevertheless, the NMR spectroscopy is mainly divided into 1D and 2D analyses. The 1D NMR analysis includes the proton (<sup>1</sup> H), carbon-13 (13C) and distortionless enhancement by polarization transfer experiment (DEPT) that provide information about the signals of protons, carbons and type of carbons (C, CH, CH2 or CH3 ) in the structure of flavonoid under elucidation. The <sup>1</sup> H NMR spectrum is very useful as it provides the number (integration value) and the type of proton involved. The chemical shift (*δ*H) values are usually exhibited within 0 (reference standard value for TMS) and 14 ppm in 1 H NMR of flavonoids while in 13C NMR, they (*δ*C) appeared between 0 and 220 ppm. Characteristic proton and carbon chemical shift values for some flavonoid classes were summarized [52, 53] (**Table 2**).

The2DNMRis composedmainlywiththeproton-protoncorrelatedspectroscopy( 1 H1 HCOSY), the heteronuclear multiple quantum coherence (HMQC)/heteronuclear single quan-tum coherence (HSQC), the heteronuclear multiple bond connectivity (HMBC), the nuclear overhauser spectroscopy (NOESY), the rotative-frame overhauser spectroscopy (ROESY) and the


**Table 2.** Characteristic proton and carbon chemical shifts for some flavonoids.

total correlated spectroscopy (TOCSY) experiments. The <sup>13</sup>C data of flavonoids in several cases could also be assigned from HMQC and HMBC spectra.

**4. Structure characterization of flavonoids**

52 Flavonoids - From Biosynthesis to Human Health

CH2

or CH3

and 14 ppm in 1

were summarized [52, 53] (**Table 2**).

**Chemical shifts (ppm) <sup>1</sup>**

**Chemical shifts (ppm) 13C** 210–170 C═O

2–3 H-3 (Flavanone), CH3

6–8 A- and B-ring protons

8–8.5 H-2 isoflavone

28–35 C-4, flavanol

**Table 2.** Characteristic proton and carbon chemical shifts for some flavonoids.

4–6 H-2 (Flavanone, dihydroflavonol)

165–155 (no ortho/para oxygenation) Oxygenated aromatic carbons 150–130 (with ortho/para oxygenation) Oxygenated aromatic carbons 135–125 (para substitution) Non-oxygenated aromatic carbons 125–90 (with ortho/para oxygenation) Non-oxygenated aromatic carbons

The structure characterization of flavonoids is related to the elucidation of their spectroscopic spectra obtained by techniques such as nuclear magnetic resonance (NMR), mass spectrometry (MS), spectrophotometric ultra-violet (UV) and infrared (IR). Physical properties of the flavonoids as melting point (mp), circular dichroism (CD), optical rotatory power ([α]D) are also useful for full characterization of the isolated flavonoid specially when its contains stereocenterfor CD and [α]D. Some chapters in this book would provide more details about the use of spectroscopic analysis to characterize flavonoids. Nevertheless, the NMR spectroscopy is mainly divided into 1D and 2D analyses. The 1D NMR analysis includes the proton (<sup>1</sup>

carbon-13 (13C) and distortionless enhancement by polarization transfer experiment (DEPT) that provide information about the signals of protons, carbons and type of carbons (C, CH,

useful as it provides the number (integration value) and the type of proton involved. The chemical shift (*δ*H) values are usually exhibited within 0 (reference standard value for TMS)

220 ppm. Characteristic proton and carbon chemical shift values for some flavonoid classes

the heteronuclear multiple quantum coherence (HMQC)/heteronuclear single quan-tum coherence (HSQC), the heteronuclear multiple bond connectivity (HMBC), the nuclear overhauser spectroscopy (NOESY), the rotative-frame overhauser spectroscopy (ROESY) and the

**H**

12–14 5-OH when C═O at C-4 (usually observed in DMSO-*d6*

80–40 Non-oxygenated (C-2, C-3 flavanone/flavanol)

H NMR of flavonoids while in 13C NMR, they (*δ*C) appeared between 0 and

) in the structure of flavonoid under elucidation. The <sup>1</sup>

The2DNMRis composedmainlywiththeproton-protoncorrelatedspectroscopy(

H),

H NMR spectrum is very

1 H1

aromatic

HCOSY),

)

The infrared spectroscopy compared to other spectroscopic techniques exhibits little but useful information in the structure characterization of flavonoids. Most of hydroxylated flavones, isoflavones and chalcones or dihydrochalcones showed maxima large band absorptions around 3300–3600 cm−1 due to hydroxyl groups. Additionally, intense band absorption characteristic for flavonoid carbonyl groups (C═O) is observed around 1680 cm−1 and is shifted approximately to 1620 cm−1 when the hydroxyl is chelated with a C═O. From the IR spectrum of flavonoids, a sharp and intense absorption band is also observed between 1600 and 1500 cm−1 due to aromatic double bonds (aromatic rings).

Ultra-violet (UV) absorption spectroscopy of flavonoids has two maxima absorptions around 300–350 and 240–285 nm corresponding to bands I and II from A- and B-rings, respectively. This technique is used for identification of the flavonoid type and its oxygenation pattern. UV-shift reagents (AlCl3 , NaOMe, NaOAc, NaOAc + H<sup>3</sup> BO<sup>3</sup> , AlCl3 + HCl) are mostly used in the sample solution to confirm the presence and the substitution pattern of hydroxyl groups in flavonoids. The presence of ortho-dihydroxylated groups could be detected by the bathochromic shift of band I after addition of NaOAc/H<sup>3</sup> BO<sup>3</sup> while the addition of AlCl3 led to the bathochromic effect of band I when the flavonoid with a carbonyl at C-4 had hydroxyl group at positions C-3 or C-5. The bathochromic shift of band II occurs especially when NaOAc is added to a solution of flavonoids having a free hydroxyl group at C-7 [3, 54, 55]. Characteristic UV absorption bands I and II due to different classes of flavonoids have been reported elsewhere (**Table 3**) [54].

The mass spectrometry technique is very helpful in the structure elucidation of flavonoids. It is used in the determination of the molecular weight for establishing the distribution of substituents between the A- and B-rings and in the determination of the nature and site of attachment of the sugar(s) in flavonoid C- and *O*-glucosides. The molecular weight of the basic


**Table 3.** Ultra-violet absorption ranges for flavonoids.

flavonoid nucleus is 222 *a.m.u*. for flavones, isoflavone and aurone; 224 *a.m.u.* for flavanones and chalcones; 238 *a.m.u.* for flavonols; and 240 *a.m.u*. for the dihydroflavonols. The molecular weight of the unknown flavonoid could be deduced by addition of atomic mass units of all its substituents [16 a.m.u (-OH), 30 a.m.u. (-OCH<sup>3</sup> ), and so on] to one of the basic molecular weights above. The loss of some ion-fragments from the molecular or pseudo-molecular ion is very characteristic in the mass spectra of flavonoids. Peaks obtained during this fragmentation process represent accurately the corresponding ion-fragments that are expressed as mass-to-charge ratio (m/z). The exact molecular weight for each fragment may be measured to the nearest 0.0001 mass unit if the mass spectrometer is operating in high resolution. This information enables calculation of precise molecular formula from the molecular ion peak and ion-fragments [54]. A prerequisite for successful mass spectrometry is that the flavonoid should be sufficiently volatile in the high vacuum within the mass spectrometer. Most aglycones are sufficiently volatile at probe temperature of 100–230°C, higher temperatures being required for the more polar polyhydroxyflavones and flavonols. Glycosides, anthocyanidins and biflavonoids, however, are not sufficiently volatile and should therefore be derivatized to improve their volatility. Some standard methods used for derivatization of compounds are permethylation or perdeuteromethylation and trimethylsilylation [54].

Natural products in general or flavonoids in particular remain an important source for drug discovery. Determination of their absolute configurations is one of the most challenging tasks in the structure elucidation of chiral flavonoids. It has been proven that the change in absolute configuration of secondary metabolites consequently affected the difference in pharmacological activity of both stereo-compounds. Methods such as chiroptical approaches, chemical synthesis, analytical chemistry, chiral derivatization and X-ray crystallography could be used to determine the absolute configuration of flavonoids. An important investigation was reported on the determination of absolute configuration of natural products and some flavonoids using experimental and calculated electronic circular dichroism (ECD) data [56].
