4. Structure identification of flavonoids

Generally, structure determination of flavonoids can be achieved easily because of the systematic research of their structures and the progress of spectroscopic technologies (Nuclear Magnetic Resonance spectroscopy, especially). Series of spectroscopic technologies, such as IR, UV, NMR, and MS, are often used during structure identification of flavonoids. In rare cases, total synthesis should be applied to verify the elucidated structures.

#### 4.1. Ultraviolet spectrum (UV)

The positions, types and number of substituents in the conjugated systems could be speculated via means of UV spectrum. Most of the flavonoids in methanol possess two main absorption bands. Band I is at 300–400 nm, which is caused by electron transition of cinnamoyl group. Band II is at 240–280 nm, which is caused by electron transition of benzoyl group, as shown in Figure 2. The structure types and oxygen-bearing substituent types of flavonoids could be determined by the peak locations, shapes and strengths of band I and II, as shown in Table 2 [27].

The locations and shapes of Band I and II will be affected by the substituents attached to rings A and B. Normally, red shift of band I increases accordingly when the number of hydroxyl groups located at ring B increases. Similarly, red shift of band II increases accordingly when the number of hydroxyl groups located at ring A increases, but it has trifling impact to band I, with the exception of 5-OH. The corresponding bands will be violet shifted 5–15 nm if the

Isolation and Structure Identification of Flavonoids http://dx.doi.org/10.5772/67810 25

Figure 2. UV spectrum of flavonoids.

3.2.2.2. High-speed counter current chromatography (HSCCC)

(9.5:10.5) two-phase system [25].

24 Flavonoids - From Biosynthesis to Human Health

respectively.

Table 2 [27].

3.2.2.3. Molecular imprinting technology (MIT)

4. Structure identification of flavonoids

4.1. Ultraviolet spectrum (UV)

synthesis should be applied to verify the elucidated structures.

High-speed counter current chromatography (HSCCC) has been applied successfully to the isolation of flavonoids. The method is simple and quick to operate, and could get product with high purity. Furthermore, it is suitable to industrial production. For example, an HSCCC system has been employed to separate seven flavonoids from a methanolic extract of the leaves of Oroxylum indicum by a one-step isocratic elution using a chloroform-methanol-water

Molecular imprinting technology (MIT) has been applied in recent years to isolation and active screening of flavonoids. As the study [26] of Pakade et al., molecularly imprinted polymers (MIPs) targeting quercetin were prepared from 4-Vinylpyridine and ethylene dimethacrylate (EDMA) under various solvent systems with the aim to form MIPs with high recognition for the quercetin molecule in aqueous systems at high temperature. The slopes for the effect of extraction time revealed that the mass transfer of the analytes was higher at 84C than at 25C. Also, the binding capacity for the most promising MIP and its corresponding NIP was higher at 84C. The binding capacity for the MIP was similar to 30 μmol/g at 25C and 120 μmol/g at 84C, while for the corresponding NIP, it was similar to 15 and 90 μmol/g, at 25 and 84C,

Generally, structure determination of flavonoids can be achieved easily because of the systematic research of their structures and the progress of spectroscopic technologies (Nuclear Magnetic Resonance spectroscopy, especially). Series of spectroscopic technologies, such as IR, UV, NMR, and MS, are often used during structure identification of flavonoids. In rare cases, total

The positions, types and number of substituents in the conjugated systems could be speculated via means of UV spectrum. Most of the flavonoids in methanol possess two main absorption bands. Band I is at 300–400 nm, which is caused by electron transition of cinnamoyl group. Band II is at 240–280 nm, which is caused by electron transition of benzoyl group, as shown in Figure 2. The structure types and oxygen-bearing substituent types of flavonoids could be determined by the peak locations, shapes and strengths of band I and II, as shown in

The locations and shapes of Band I and II will be affected by the substituents attached to rings A and B. Normally, red shift of band I increases accordingly when the number of hydroxyl groups located at ring B increases. Similarly, red shift of band II increases accordingly when the number of hydroxyl groups located at ring A increases, but it has trifling impact to band I, with the exception of 5-OH. The corresponding bands will be violet shifted 5–15 nm if the


Table 2. The spectral characteristics of UV-VIS spectrum of flavonoids.

particular hydroxyl is glycosided. Furthermore, the influence of the hydroxyl groups will almost disappear if they are acetylated.

#### 4.2. Infrared spectrum (IR)

It is used mainly to determine the types of functional groups, substitution modes of aromatic rings and so on. The all functional groups, such as carbonyl, phenolic hydroxyl, phenyl and glycosyl, have possessed corresponding IR absorptions. The absorption band of hydroxyl groups are in the 3200–3650 cm<sup>1</sup> region, carbonyl groups are in 1660–1680 cm<sup>1</sup> region and the vibrations of benzene rings are at about 1500, 1580 and 1600 cm<sup>1</sup> .

#### 4.3. Nuclear magnetic resonance spectrum (NMR)

Nuclear magnetic resonance spectrum (NMR) is the most powerful method to elucidate the structures of flavonoids. Kinds of solvents, such as CDCl3, DMSO-d6, C5D5N, (CD3)2CO and CD3OD, could be employed while performing NMR experiments. DMSO-d<sup>6</sup> is the optional solvent among them to perform NMR to flavonoids. Almost all kinds of flavonoids could be well dissolved in DMSO-d6, and the resonance signals of flavonoids are rarely overlapped by solvent peaks (about δ2.5). Furthermore, NMR signals of phenolic hydroxyl groups could be displayed clearly with DMSO-d<sup>6</sup> as the solvent. The drawback of this solvent is high boiling point, which leads to difficulty in sample recovery.

#### 4.3.1. <sup>1</sup> H-NMR spectrum

It provides information of chemical shifts, coupling constants and proton number. The types of flavonoids, substituted modes, number and configurations of glycosyls and so on, could be determined via <sup>1</sup> H-NMR spectrum.

#### 4.3.1.1. Protons on ring C

1 H-NMR characteristics of protons on ring are shown in Table 3 [28].

#### 4.3.1.2. Protons on ring A

The ordinary substitution modes are 5,7-dioxygenation, 7-oxygenation, 5,6,7-trioxygenation and 5,7,8-trioxygenation, See Figure 3.

#### 4.3.1.2.1. 5,7-Dihydroxyl substituted

5,7-Dihydroxyl flavonoids are most common. For this type of flavonoids, the signals of H-6 and H-8 are shown at δ5.7–6.9 as doublets, and the signal of H-6 is always at the higher field than H-8. The signals of both H-6 and H-8 shift to lower field after glycosidation of 7-OH.


Table 3. Chemical shifts and coupling constants of ring C of common flavonoids.

Isolation and Structure Identification of Flavonoids http://dx.doi.org/10.5772/67810 27

Figure 3. Substitution modes of ring A.

well dissolved in DMSO-d6, and the resonance signals of flavonoids are rarely overlapped by solvent peaks (about δ2.5). Furthermore, NMR signals of phenolic hydroxyl groups could be displayed clearly with DMSO-d<sup>6</sup> as the solvent. The drawback of this solvent is high boiling

It provides information of chemical shifts, coupling constants and proton number. The types of flavonoids, substituted modes, number and configurations of glycosyls and so on, could be

The ordinary substitution modes are 5,7-dioxygenation, 7-oxygenation, 5,6,7-trioxygenation

5,7-Dihydroxyl flavonoids are most common. For this type of flavonoids, the signals of H-6 and H-8 are shown at δ5.7–6.9 as doublets, and the signal of H-6 is always at the higher field than H-8. The signals of both H-6 and H-8 shift to lower field after glycosidation of 7-OH.

Flavanone δ6.3– 6.8 (s) The signals maybe overlapped by H-6 or H-8.

(dd, J¼17, 11 Hz); (dd, J ¼ 17, 5 Hz)

Hz)

Table 3. Chemical shifts and coupling constants of ring C of common flavonoids.

Isoflavone δ7.6–7.8 (s) The signal is at rather low field because of influence of

δ5.0–5.6 δ4.3–4.6 After glycosidation of 3-OH, resonance signals of both

oxygen atom at position 1 and carbonyl at position 4.

C-2 and C-3 form a trans double bond.

H-2 is coupled by two protons of position 3.

Configurations of both C-2 and C-3 are R.

H-2 and H-3 shift to low field.

point, which leads to difficulty in sample recovery.

H-NMR spectrum.

Type 2-H 3-H Note

H-NMR characteristics of protons on ring are shown in Table 3 [28].

H-NMR spectrum

26 Flavonoids - From Biosynthesis to Human Health

4.3.1. <sup>1</sup>

1

determined via <sup>1</sup>

4.3.1.1. Protons on ring C

4.3.1.2. Protons on ring A

Flavonol None signal.

Flavanone-3-Oglycoside

Aurone Exocyclic proton:

δ6.5–6.7 (s)

Chalcone α-H: δ6.7–7.4 (d, J ¼ 17 Hz)

β-H: δ7.3–7.7 (d, J ¼ 17 Hz)

Flavanone δ5.0–5.5 (dd, J ¼ 11, 5 Hz) δ2.3– 2.8 (2H)

Flavanonol 4.8–5.0 (d, J ¼ 11 Hz) δ4.1–4.3 (d, J ¼ 11

and 5,7,8-trioxygenation, See Figure 3.

4.3.1.2.1. 5,7-Dihydroxyl substituted

#### 4.3.1.2.2. 7-Hydoxyl substituted

Signal of H-5 is shown to be a doublet since vicinal coupling exists between H-5 and H-6. Additionally, the chemical shift is at rather low field (about δ8.0) because of the shielding effect of carbonyl at position 4. H-6 is affected by H-5 and H-8, so it has showed a double-doublet (dd, J¼2.0, 8.0 Hz). H-8 is showed to be a doublet (J¼2.0Hz) because of the vicinal relationship with H-6. Signals of both, H-6 and H-8 are at δ6.3–7.1. The chemical shifts of protons on ring A are shown in Table 4 [28].

#### 4.3.1.3. Protons on ring B

There are avarietyofsubstitutedmodesof ringB,suchasnon-substitution,4'-oxygenation, 2'-oxygenation, 3',4'-dioxygenation, 2',4'-oxygenation, 3',4',5'-trioxygenation and 2',4',5'-trioxygenation, as shown in Figure 4. Generally, signals of protons on ring B are showed at slightly lower field, and the chemical shifts are usual at δ6.7-8.1. The substitution modes and structural information could be determinedvia the chemical shifts and coupling constants of ringB.

#### 4.3.1.3.1. None substituent on ring B

For this mode, there are five protons on ring B. Signals of H-2' and H-6' are shown at lower field than H-3', H-4' and H-5' because of the shielding effect of ring C. Furthermore, the peak shapes of all of the protons are complicated because of the coupling effects of the vicinal- and


Table 4. Chemical shifts of protons on ring A.

Figure 4. Substitution modes of ring B.

meta-coupling. The signals of H-2' and H-6' are usually at δ7.1–7.6 and of H-3<sup>0</sup> , H-4<sup>0</sup> and H-5<sup>0</sup> are at δ7.9–8.2.

#### 4.3.1.3.2. 4<sup>0</sup> -Oxygenation

In this circumstance, ring B is a symmetrical substructure. One AA'BB' coupling system is formed by four aromatic protons. The spectral characteristics are show in Table 5 [28].

#### 4.3.1.3.3. 3<sup>0</sup> ,40 -Dioxygenation

In this circumstance, one ABX coupling system is formed by three aromatic protons, and three groups of signals are displayed as H-2<sup>0</sup> (1H, d, J ≈ 2.0Hz), H-5<sup>0</sup> (1H, d, J ≈ 8.0Hz) and H-6<sup>0</sup> (1H, dd, J ≈ 2.0, 8.0Hz). The chemical shifts of protons on ring B are shown in Table 6 [28].


Table 5. Chemical shifts of protons on ring B of 4<sup>0</sup> -oxygenated flavonoids.


Table 6. Chemical shifts of protons on ring B of 3<sup>0</sup> ,40 -dioxygenated flavonoids.

#### 4.3.1.3.4. 2<sup>0</sup> -Oxygenation

ABCD coupling system is formed by the rest protons of ring B. The peak shapes are rather complicated. Signals of H-3' and H-5' are usually displayed at δ6.8–6.9, H-4' at about δ7.2 and H-6' at δ7.4–7.5.

#### 4.3.1.3.5. 3<sup>0</sup> ,40 ,50 -Trioxygenation

meta-coupling. The signals of H-2' and H-6' are usually at δ7.1–7.6 and of H-3<sup>0</sup>

In this circumstance, ring B is a symmetrical substructure. One AA'BB' coupling system is

In this circumstance, one ABX coupling system is formed by three aromatic protons, and three

(1H, d, J ≈ 2.0Hz), H-5<sup>0</sup>


formed by four aromatic protons. The spectral characteristics are show in Table 5 [28].

dd, J ≈ 2.0, 8.0Hz). The chemical shifts of protons on ring B are shown in Table 6 [28].

are at δ7.9–8.2.


Figure 4. Substitution modes of ring B.

28 Flavonoids - From Biosynthesis to Human Health


groups of signals are displayed as H-2<sup>0</sup>

Table 5. Chemical shifts of protons on ring B of 4<sup>0</sup>

Type H-2<sup>0</sup>

Flavanone δ7.1–7.3

Flavanonol δ7.2–7.4 Isoflavone δ7.2–7.5 Chalcone δ7.4–7.6 Aurone δ7.6–7.8 Flavone δ7.7–7.9 Flavonol δ7.9–8.1

4.3.1.3.2. 4<sup>0</sup>

4.3.1.3.3. 3<sup>0</sup>

,40

, H-4<sup>0</sup> and H-5<sup>0</sup>

(1H,

, H-5<sup>0</sup>

δ6.5–7.1

(1H, d, J ≈ 8.0Hz) and H-6<sup>0</sup>

, H-6<sup>0</sup> H-3<sup>0</sup>

If identical substituents are attached to C-3' and C-5', which allows the formation of a symmetrical substructure of ring B, H-2' and H-6' will display to be a singlet at δ6.5–7.5.

#### 4.3.1.3.6. 2<sup>0</sup> ,40 ,50 -Trioxygenation

In the cases of this substituent mode, either of the two protons on ring B displays to be a singlet. Generally, signals of H-6' in flavones and flavonols are showed at δ7.2–7.5, H-3' at δ6.4–6.6. Signals of H-6' are shown at slight higher field.

#### 4.3.1.3.7. 2<sup>0</sup> ,40 -Dioxygenation

In the cases of this mode, H-3<sup>0</sup> will be showed at δ6.00–6.6 (d, J ≈ 2.0 Hz), H-5<sup>0</sup> at δ 6.6–6.5 (dd, J ≈ 2.0, 8.0 Hz) and H-6<sup>0</sup> at δ7.0–7.4 (d, J ≈ 8.0 Hz). See Table 7.

#### 4.3.1.4. Common substituents

The proton chemical shifts of common substituents of flavonoids are shown in Table 8 [28].

#### 4.3.2. 13C-NMR spectrum

Strong regularities are also shown in 13C-NMR spectra of flavonoids. The types of flavonoids, number and connection positions of glycosyls could be elucidated from 13C-NMR spectra.

#### 4.3.2.1. Identification of skeleton structures of flavonoids

The core structures are difficult to be elucidated by resonance signals of aromatic protons. However, the characteristic signals of carbons in ring C allowed the identification of different types of flavonoids, see Table 9.


Table 7. Chemical shifts of protons on ring B of various substituent modes [28].


Table 8. Chemical shifts of the protons on common substituents.

#### 4.3.2.2. Determination of substituent modes of flavonoids

The substituent modes of core structures of flavonoids could be determined by the signals of aromatic carbons. The chemical shifts of carbons in ring A and B, if they are not substituent, are shown in Table 10 [28].

#### 4.3.2.2.1. Signal characteristics of ring A

Usually, the substituents, such as hydroxyl, methoxyl and isopentenyl groups, are attached at position 5 or/and 7 of ring A, which leads to the changes of chemical shifts of other carbons in ring A. It is shown in Table 11 [28].

#### 4.3.2.2.2. Signal characteristics of carbons on ring B

The signal characteristics of carbons on ring B are shown in Table 12 [28].

#### Isolation and Structure Identification of Flavonoids http://dx.doi.org/10.5772/67810 31


Table 9. Chemical shifts of carbons in ring C of flavonoids [28].


Table 10. Chemical shifts of carbons in ring A and B if they are not substituent.


Table 11. Chemical shifts of carbons in ring A of flavonoids.

4.3.2.2. Determination of substituent modes of flavonoids

Table 8. Chemical shifts of the protons on common substituents.

shown in Table 10 [28].

20

40

20 ,40

30 ,40

30 ,40 ,50

20 ,40 ,50


30 Flavonoids - From Biosynthesis to Human Health



30 ,4<sup>0</sup> and 5<sup>0</sup> .

Proton type Chemical shift

Methoxyl δ3.5–4.1 (3H,s)

O-CH2-O δ6.0

Terminal protons of glycosyl δ4.5–5.5

J≈8.0Hz)

J≈2.0Hz)

Table 7. Chemical shifts of protons on ring B of various substituent modes [28].

Phenolic hydroxyl 5-OH (δ12.0–14.0),7-OH (δ10.8–11.0), 4<sup>0</sup>

Isopentenyl δCH2 (3–3.4); CH (5.2);CH3 (δ1.7–1.8)

Methyl C-6,8 (δ2.0–2.5); rha-CH3 [δ9.2–10.4 (d, J≈6.5Hz)]


4.3.2.2.1. Signal characteristics of ring A

ring A. It is shown in Table 11 [28].

4.3.2.2.2. Signal characteristics of carbons on ring B

The signal characteristics of carbons on ring B are shown in Table 12 [28].

The substituent modes of core structures of flavonoids could be determined by the signals of aromatic carbons. The chemical shifts of carbons in ring A and B, if they are not substituent, are

Substituent mode H-2<sup>0</sup> H-3<sup>0</sup> H-4<sup>0</sup> H-5<sup>0</sup> H-6<sup>0</sup> None substituent on ring B δ7.1–7.6 (m) δ7.9–8.2 (m) δ7.9–8.2 (m) δ7.9–8.2 (m) 7.1-7.6 (m)

> δ6.5–7.1 (2H, d, J≈8.0Hz)

J≈2.0Hz)

,60



δ6.5–7.1 (2H, d, J≈8.0Hz)

δ6.30–6.50 (1H, dd, J≈8.0,2.0Hz)

δ6.7–7.1(1H, d, J≈8.0Hz)


) as identical oxygen-bearing substituents are connected to position

CH3CO [glc: δ1.65–2.10 (3H,s); aromatic -CH3:δ2.3–2.5 (3H,s)

7.1–8.1 (2H, d, J≈8.0Hz)

7.0–7.4(d, J≈8.0Hz)


6.7–7.9(1H, dd, J≈2.0, 8.0Hz)

Usually, the substituents, such as hydroxyl, methoxyl and isopentenyl groups, are attached at position 5 or/and 7 of ring A, which leads to the changes of chemical shifts of other carbons in

#### 4.3.2.2.3. Signal characteristics of common substituents

The carbon chemical shifts of common substituents are shown in Table 13 [28].


Table 12. Chemicals shifts of carbons on ring B of flavonoids.

#### 4.3.3. Glycosides of flavonoids

In plants, flavonoids are often present as O- or C-glycosides. The O-glycosides have sugar substituents bound to a hydroxyl group of the aglycone, usually located at position 3 or 7, whereas the C-glycosides have sugar groups bound to a carbon of the aglycone, usually 6-C or 8-C. The most common carbohydrates are rhamnose, glucose, galactose and arabinose.

Generally, the chemical shifts of terminal protons of glycosyls are at δ4.5–5.5 in <sup>1</sup> H-NMR. The terminal carbons of O-glycosides are at δ95–105 and at δ71–78 for C-glycosides. Furthermore, the number of glycosyls could be determined by combined analysis of <sup>1</sup> H and 13C-NMR spectra. It is an effective method to determine the connection positions of glycosyls by glycosylation shifts, as shown in Table 14 [28].

The configurations of glycosyls should be determined. The relative configurations of some glycosyl groups could be determined sometimes by coupling constants of terminal protons in 1 H-NMR spectra. The absolute configurations, however, should be determined by chemical methods and gas chromatography.


Table 13. Chemical shifts of carbons of common substituents on flavonoids.


Table 14. Glycosylation shifts (average values) of flavonoids in 13C-NMR spectrum.

As for the spectral method, the types and configurations could be speculated by the chemical shifts of glycosyl carbons in 13C-NMR spectra, as shown in Table 15 [28].

#### 4.4. Mass spectral characteristics of flavonoids

4.3.3. Glycosides of flavonoids

1

30 -OH,4<sup>0</sup>

30 -OCH3,4<sup>0</sup>

30 -OH,4<sup>0</sup>

30 -OCH3,4<sup>0</sup>

30 ,40 ,50 - Trioxygenation

(or 3<sup>0</sup> ,40

(or 3<sup>0</sup> ,40 -OCH3



32 Flavonoids - From Biosynthesis to Human Health


sylation shifts, as shown in Table 14 [28].

Table 12. Chemicals shifts of carbons on ring B of flavonoids.

methods and gas chromatography.

In plants, flavonoids are often present as O- or C-glycosides. The O-glycosides have sugar substituents bound to a hydroxyl group of the aglycone, usually located at position 3 or 7, whereas the C-glycosides have sugar groups bound to a carbon of the aglycone, usually 6-C or 8-C. The most common carbohydrates are rhamnose, glucose, galactose and arabinose.

Substituent mode Type C-1<sup>0</sup> C-2<sup>0</sup> C-3<sup>0</sup> C-4<sup>0</sup> C-5<sup>0</sup> C-6<sup>0</sup> 4'-Oxygenated Flavone, flavonol, isoflavone δ121–123 δ130 δ115 δ157–161 δ115 δ130

Flavone, flavonol, isoflavone δ121–125 δ113–114 δ145–147 δ149–151 δ112–116 δ118–122

Flavanone, flavanonol δ128–129 δ111–115 δ144–147 δ146–148 δ112–116 δ118–120

δ120–126 δ106–109 δ146–153 δ93–97 δ136–142 δ106–109



2',4'-Dioxygenation Flavonoids δ108–113 δ156–158 δ102–104 δ157–162 δ104–108 δ131–132

Flavanone, flavanonol δ128–130

terminal carbons of O-glycosides are at δ95–105 and at δ71–78 for C-glycosides. Furthermore,

spectra. It is an effective method to determine the connection positions of glycosyls by glyco-

The configurations of glycosyls should be determined. The relative configurations of some glycosyl groups could be determined sometimes by coupling constants of terminal protons in

H-NMR spectra. The absolute configurations, however, should be determined by chemical

Isopentenyl [-CH2CH¼CHCH3] CH2(δ21–22); CH(δ122–124; CH(δ129–131); CH3(δ17–27)

CH3 6-CH3(δ6–10);8-CH3(δ20–30);COCH3(δ17–22)

C-glycosides δ71–80

H-NMR. The

H and 13C-NMR

Generally, the chemical shifts of terminal protons of glycosyls are at δ4.5–5.5 in <sup>1</sup>

the number of glycosyls could be determined by combined analysis of <sup>1</sup>

Substituent Chemical shift

OCH3 δ55–57

O-CH2-O δ100–101 Terminal carbon of glycosyls O-glycosides δ95–105

Table 13. Chemical shifts of carbons of common substituents on flavonoids.

ESI-MS and FAB-MS are widely applied in the studies of flavonoids. While the positive ion mode is employed, quasi-molecular ion peaks such as [MþH]þ, [MþNa]þ, [MþK]<sup>þ</sup> and [MþNH4] <sup>þ</sup> will be displayed. [2MþH]þ, [2MþNa]<sup>þ</sup> and so on will also be shown if the sample is concentrated. The MS fragmentation pathways of flavone and flavanone are shown in Figures 5 and 6.

#### 4.5. Determination of absolute configuration

The absolute configuration should be determined if chiral atoms are existed in the structures. The main methods to elucidate absolute configuration include circular dichroism (CD), optical rotatory dispersion (ORD) and X-ray single crystal diffraction. Circular dichroism and ORD are mainly introduced here.


Table 15. Carbon chemical shifts of common glycosyls.

Figure 5. MS fragmentation pathway of flavone.

#### 4.5.1. Optical rotatory dispersion (ORD)

For the flavonoids possess chiral centers, their optical activities (589.0 nm, Na-D light source) are correlative with spatial configurations, as shown in Table 16 [27].

#### 4.5.2. Circular dichroism (CD)

It is the most used method to elucidate the absolute configurations of flavonoids via cotton effect (CE) of CD spectra.

#### 4.5.2.1. Flavanone

Most of the protons of flavanones at position 2 are axial (J ≈ 11.0Hz). The characteristics of CE are shown in Table 17.

As reported in literature [29], the absolute configurations of the enantiomeric flavanone pair (2S)-6-formyl-5,7-dihydroxyflavanone (1a) and (2R)-6-formyl-5,7-dihydroxyflavanone (1b) were assessed via their chiroptical data. The ECD curves of compound (1a) showed sequential positive and negative cotton effects near 310 and 280 nm for the n!π\* and π!π\* electronic transitions, respectively. These cotton effects are reminiscent of flavanones exhibiting

Isolation and Structure Identification of Flavonoids http://dx.doi.org/10.5772/67810 35



Table 16. Optical activities of flavonoids.

4.5.1. Optical rotatory dispersion (ORD)

Figure 5. MS fragmentation pathway of flavone.

34 Flavonoids - From Biosynthesis to Human Health

4.5.2. Circular dichroism (CD)

effect (CE) of CD spectra.

are shown in Table 17.

4.5.2.1. Flavanone

For the flavonoids possess chiral centers, their optical activities (589.0 nm, Na-D light source)

It is the most used method to elucidate the absolute configurations of flavonoids via cotton

Most of the protons of flavanones at position 2 are axial (J ≈ 11.0Hz). The characteristics of CE

As reported in literature [29], the absolute configurations of the enantiomeric flavanone pair (2S)-6-formyl-5,7-dihydroxyflavanone (1a) and (2R)-6-formyl-5,7-dihydroxyflavanone (1b) were assessed via their chiroptical data. The ECD curves of compound (1a) showed sequential positive and negative cotton effects near 310 and 280 nm for the n!π\* and π!π\* electronic transitions, respectively. These cotton effects are reminiscent of flavanones exhibiting

are correlative with spatial configurations, as shown in Table 16 [27].


Table 17. Relationship between CE and absolute configurations of flavanones.

P-helicity of the conformational flexible heterocycle with a C-2 equatorial B ring and, hence, (2S) absolute configuration. The mirror image related to ECD spectrum of 1b accordingly confirmed its (2R) absolute configuration. It is shown in Figure 7.

Figure 7. ECD spectra for compounds 1a and 1b.

#### 4.5.2.2. Flavanonol

Four possible structures are existed in nature because of the existence of two chiral centers (C-2 and C-3), while 2R, 3R configurations are commonest. The relative configuration could be determined by coupling constant between H-2 and H-3 and then CD spectrum is employed to elucidate the absolute configuration, as shown in Table 18 [30].

#### 4.5.2.3. 3-Hydroxyl flavans

Similarly, C-2 and C-3 are also the chiral centers of 3-hydroxyl flavans. The characteristics are shown in Table 19 [31].

#### 4.5.2.4. 4-Hydroxyl flavans

The relative configuration could be determined by coupling constant of H-2 and H-4 combined with NOE spectra and then CD spectrum could be employed to elucidate the absolute configuration, as shown in Table 20 [32].


Table 18. Relationship between absolute configurations of flavanonol with CE.


Table 19. Relationship between absolute configurations of 3-hydroxyl flavans with CE.


Table 20. Relationship between absolute configurations of 4-hydroxyl flavans with CE.


Table 21. Relationship of absolute configurations of 3,4-dihydroxyl flavans with CE.

#### 4.5.2.5. 3,4-Dihydroxyl flavans

More absolute configurations are existed because of three chiral centers, as shown in Table 21 [33].

#### 4.5.2.6. Flavans

P-helicity of the conformational flexible heterocycle with a C-2 equatorial B ring and, hence, (2S) absolute configuration. The mirror image related to ECD spectrum of 1b accordingly

Four possible structures are existed in nature because of the existence of two chiral centers (C-2 and C-3), while 2R, 3R configurations are commonest. The relative configuration could be determined by coupling constant between H-2 and H-3 and then CD spectrum is employed

Similarly, C-2 and C-3 are also the chiral centers of 3-hydroxyl flavans. The characteristics are

The relative configuration could be determined by coupling constant of H-2 and H-4 combined with NOE spectra and then CD spectrum could be employed to elucidate the absolute config-

Relative configuration Cotton effect (300–340) Absolute configuration

� 2S, 3S

� 2S, 3R

trans- þ 2R, 3R

cis- þ 2R, 3S

Table 18. Relationship between absolute configurations of flavanonol with CE.

confirmed its (2R) absolute configuration. It is shown in Figure 7.

to elucidate the absolute configuration, as shown in Table 18 [30].

4.5.2.2. Flavanonol

Figure 7. ECD spectra for compounds 1a and 1b.

36 Flavonoids - From Biosynthesis to Human Health

4.5.2.3. 3-Hydroxyl flavans

shown in Table 19 [31].

4.5.2.4. 4-Hydroxyl flavans

uration, as shown in Table 20 [32].

The cotton effects of flavans are show in Table 22 [34].

#### 4.5.2.7. Isoflavans

#### The CE characteristics are shown in Table 23.


Table 22. Cotton effects of flavans.


Table 23. Cotton effects of isoflavans.


Versteeg et al. [35] synthesized six isoflavans and their enantiomers (31a/b-36a/b), and used authentic 3S- and 3R-vestitol (30a and 30b) derivatives to establish the absolute configuration at C3 of the synthetic isoflavans (Figure 8). (3S)-Isoflavans with oxygenation at both the A- and B-rings (34a, 35a and 36a) display positive and negative CEs in the 240 (1La) and 270– 280 nm (1Lb) regions, respectively, and conversely for the 3R-enantiomers (34b, 35b and 36b) (Figure 9).

Figure 9. CD spectra of isoflavans with oxygenation at both the A and B rings.

4.5.2.7. Isoflavans

Table 22. Cotton effects of flavans.

Table 23. Cotton effects of isoflavans.

Figure 8. Synthetic isoflavans (31a/b–36a/b).

The CE characteristics are shown in Table 23.

38 Flavonoids - From Biosynthesis to Human Health

260–320 nm 220–260 nm

Cotton effect (280 nm) Absolute configuration

CE Absolute configuration

þ 2R � 2S

þ � 3R � þ 3S


Table 24. Relationship of absolute configurations of pterocarpins with CE.

#### 4.5.2.8. Pterocarpins

The spectral characteristics are shown in Table 24 [36].

The relationships between the CE and absolute configurations will change after a hydroxyl group is attached to position 6a, as shown in Table 25 [37].


Table 25. Relationship of absolute configurations of 6a-hydroxyl pterocarpins with CE.
