**3. Spectroscopic identification**

Asia have much lower risk of developing prostate cancer compared to Americans due to high consumption of soy rich in isoflavonoids. Upon immigration to the USA and changing the dietary components, this difference rapidly disappears [12]. Isoflavonoids are also classified as dietary antioxidants [13]. These facts were the driving force behind the use of isoflavonoid-

Isoflavonoids are a large subclass of the most common plant polyphenols containing 15 carbon atoms known as flavonoids [15]. In isoflavonoids (3-phenylchromans), the phenyl ring B is attached to heterocyclic ring C at position 3 rather than 2 in flavonoids [16]. Generally, flavonoids are biosynthesised via Shikimic acid pathway. Shikimic acid is also a precursor for the biosynthesis of phenylpropanoids and aromatic acids. At certain stages, the activity of the key enzyme chalcone isomerase (CHI) resulted in the formation of flavanones that converted to isoflavonoids under the influence of isoflavone synthase [17]. The biosynthesis of isoflavonoids, consequently, is considered as an offshoot from the flavonoids biosynthetic pathway [18]. Highest level of isoflavonoids occurs usually in roots, seedlings and seeds [18, 19].

Isoflavonoids are sub-classified into many subclasses based on the oxidation status of ring C as well as the formation of a forth ring 'D' by coupling between rings B and C. Subclasses free from ring D include isoflavones, isoflavanones, isoflavan-4-ol, homoisoflavonoids, isoflavans and isoflav-3-ene. Rotenoids, pterocarpans, coumaronochromones and coumaronochromene

This chapter will deal with the different aspects of the isoflavonoid subclasses keeping the original three-ring skeleton (**Figure 1**). Occurrence, isolation, key spectroscopic characters

The most popular method used for extraction of isoflavonoids is maceration with either MeOH

O at room temperature followed by liquid-liquid

rich sources as nutraceutical and dietary supplements [14].

62 Flavonoids - From Biosynthesis to Human Health

represent the subclasses with additional ring D formation [11].

and biological activities will be covered starting from 2000 to date.

**2. Extraction and purification**

or EtOH containing various percentages of H2

**Figure 1.** The skeletons of the isoflavonoids with three-ring structures.

#### **3.1. Infrared (IR) transmission spectra**

Both phenolic hydroxyls and carbonyl groups are present in most of the isoflavonoid classes. However, the most characteristic feature of isoflavans and isoflavenes is the lack of carbonyl function bands. The absorption bands for the C-4 carbonyl in isoflavones and isoflavanones present in the range 1606–1694 cm−1 [9, 23–26]. Differentiation between isoflavones and isoflavanones from the position of C-4 carbonyl bands in the IR spectra is not achievable.

#### **3.2. Ultra Violet (UV) absorption spectra**

In spite of the tremendous advances in 2D-NMR and MS, the UV absorption spectra in MeOH and MeOH with shift reagent still can provide useful information for flavonoids identification. In all isoflavonoids except isoflavenes, ring B has no or little conjugation with the main chromophore composed of rings A and C. This fact is expressed as intense band II and diminished band I [60].

For isoflavones, band II shows absorption at λmax 245–275 nm. Shift reagents can be used to detect hydroxylation at ring A. NaOAc induces 6–20 nm bathochromic shift as an indication of free 7-hydroxyl group. The 10–14 nm shift with AlCl3 /HCl is diagnostic for free 5-OH group. The absence of any shift with NaOMe is an evidence for the absence of free hydroxyls in ring A [19, 27, 28, 50, 60].

The UV spectra of about 28 published isoflavanone were reviewed. Band II absorption was found in the range 270–295 nm [5, 9, 23, 25, 29, 33, 39, 41, 43, 44, 47–50, 55, 61, 62]. Among these publications, only three used shift reagents with five isolated isoflavanones. Analysis of the obtained results revealed that AlCl3 induced 17–23 nm bathochromic shift in band II due to the complex formed between C-4 carbonyl and C-OH groups. All the entitled compounds contain C-7 free hydroxyl groups, and NaOAc produced 34–37 nm bathochromic shift in band II [39, 47, 50]. However, more data are required to draw a solid conclusion.

The few available UV data of homoisoflavonoids showed band II absorption in the same range reported for isoflavanones [63].

Isoflavans UV spectra show one prominent maxima representing band II between 270 and 295 nm [21, 37, 38, 45]. The available UV data of isoflavenes indicated the presence of two bands at 235–245 and 320–337 nm along with a shoulder 287–300 nm [29, 30, 31, 35, 36].

#### **3.3. Circular Dichroism (CD) Spectroscopy**

Saturation of the double bond between C-2 and C-3 creates a new asymmetric center in the molecules. The orientation at these centers is in most cases determined from the CD spectra.

Isoflavanones show three absorption maxima at 200–240, 260–300 and 320–352 nm. Determination of the absolute configuration at C-3 is based on the n→π\* carbonyl transition between 320 and 352 nm. The positive sign at this region is diagnostic for (3*R*) orientation with ring B having equatorial position. The coupling constant between the *trans*-diaxial H2β and H3 can confirm the equatorial orientation of ring B [64]. Optical inactivity of isoflavanones most probably is a result of racemization that can occur during extraction and purification [64]. The isolation of two racemic mixtures, 3*S*- and 3*R*-7-O-glucosyldiphysolones (**2, 3**) and (3*S*)- and (3*R*)-7,4′-di-O-glucosyldiphysolones (**4, 5**), from *Ormocarpum kirkii* was explained as result of isomerization in aqueous solution [32]. The same observation was reported in three isolated isoflavanones from *Platycelphium voënse* and *Desmodium canum* [41, 47]. Due to the positive cotton effect at 337 nm, the (3*R*) orientation was assigned to eryzerin B (**6**). However, eryzerin A (**7**) was reported in the same publication with undetermined absolute stereochemistry [44]. The (3*R*) orientation was also assigned to 2,3-dihydro-7-demethylrobustigenin (**8**) and saclenone (**9**) isolated from *Erythrina sacleuxii* based on the positive cotton effect at 320 and 334 nm, respectively [49].

Isoflavans configuration is much more complicated. The heterocyclic ring C is expected to have the half-chair form a fact that can be diagnosed from the vicinal coupling constants between H-2, H-3 and H-4 protons. Such *J* values along with the CD curves can then lead to determination of the absolute configuration [64]. (3*S*)-isoflavans with oxygenation at both the A and B rings display positive and negative cotton effects at 240 and 270–280 nm regions, respectively. The opposite was observed for the (3*R*)-enantiomers. The 7-deoxy (3*S*)-isoflavans with mono- and di-oxygenation at ring B displayed negative cotton effects in both the 230–240 and 270–290 nm regions, and the opposite was observed for the (3*R*)-enantiomers [64]. The difficulty in assigning the absolute configuration of isofalvans was reflected by Bedane et al. [37]. The authors isolated two new isoflavans, erylivingstone J (**10**) and erylivingstone K (**11**). The measured CD spectrum showed negative cotton effect near 306 nm and a positive cotton effect near 240 nm supporting (*S*)-configuration. Three known compounds, 2′-methoxyphaseollinisoflavan (**12**), 7,4′-dihydroxy-2′,5-dimethoxy isoflavan (**13**) and 7,4′-dihydroxy-2′-methoxy-3′- (3-methylbut-2-enyl) isoflavan (**14**), with (*R*)-absolute configuration were isolated from the same source in this study. Suspicions about the purity of the new compounds and isolation of compounds with (*R*)-absolute configuration led the authors to report the new compounds without absolute configuration [37]. The enantiomer (3*S*) (+) 2′-*O*-methylphaseollidinisoflavan (**15**) was isolated from *Erythrina caffra* along with the (3*R*) (−) erythbidin A (**16**). The configuration was assigned based on 1 H-NMR *J* values, optical rotation and CD spectra. However, the reported CD data did not cover the lower range of the spectrum near 240 nm [45]. The absolute configuration of abruquinone L (**17**) was successfully assigned by combination of 1 H-NMR analyses of the *J* values between ring C protons and the CD spectrum which showed a strong positive cotton effect at 202 nm and two negative cotton effects at 212 and 233 nm [38]. Due to the positive cotton effect at 337 nm, the (3*R*) orientation was assigned to eryzerin C (**18**). However, eryzerin D (**19**) was reported with undetermined absolute stereochemistry [44].

In case of isoflavan-4-ol, C-4 becomes a new chiral center and 4 isomers could exist. Out of the possible isomers, two are *cis-* and two are *trans-*. Hata et al. synthesized and compared the CD spectra of four stereoisomers. The 3*R*, 4*S*-*trans*-isoflavan-4-ol stereoisomer showed negative cotton effect between 250 and 300 nm and positive cotton effect between 220 and 240 nm. The other 3*S*, 4*R*-*trans*-isoflavan-4-ol stereoisomer showed CD spectrum having cotton effect at the same ranges but with opposite sign. The 3*S*, 4*S cis*-isoflavan-4-ol stereoisomer expressed positive cotton effect between 245 and 300 nm, while the other enantiomer 3*R*, 4*R*-isoflavan-4-ol has a negative cotton effect at the same region [65].

#### **3.4. Nuclear Magnetic Resonance (NMR) Spectroscopy**

#### *3.4.1. 1 H- and 13C-NMR*

For isoflavones, band II shows absorption at λmax 245–275 nm. Shift reagents can be used to detect hydroxylation at ring A. NaOAc induces 6–20 nm bathochromic shift as an indica-

group. The absence of any shift with NaOMe is an evidence for the absence of free hydroxyls

The UV spectra of about 28 published isoflavanone were reviewed. Band II absorption was found in the range 270–295 nm [5, 9, 23, 25, 29, 33, 39, 41, 43, 44, 47–50, 55, 61, 62]. Among these publications, only three used shift reagents with five isolated isoflavanones. Analysis of

to the complex formed between C-4 carbonyl and C-OH groups. All the entitled compounds contain C-7 free hydroxyl groups, and NaOAc produced 34–37 nm bathochromic shift in

The few available UV data of homoisoflavonoids showed band II absorption in the same

Isoflavans UV spectra show one prominent maxima representing band II between 270 and 295 nm [21, 37, 38, 45]. The available UV data of isoflavenes indicated the presence of two bands at

Saturation of the double bond between C-2 and C-3 creates a new asymmetric center in the molecules. The orientation at these centers is in most cases determined from the CD spectra. Isoflavanones show three absorption maxima at 200–240, 260–300 and 320–352 nm. Determination of the absolute configuration at C-3 is based on the n→π\* carbonyl transition between 320 and 352 nm. The positive sign at this region is diagnostic for (3*R*) orientation with ring B having equatorial position. The coupling constant between the *trans*-diaxial H2β and H3 can confirm the equatorial orientation of ring B [64]. Optical inactivity of isoflavanones most probably is a result of racemization that can occur during extraction and purification [64]. The isolation of two racemic mixtures, 3*S*- and 3*R*-7-O-glucosyldiphysolones (**2, 3**) and (3*S*)- and (3*R*)-7,4′-di-O-glucosyldiphysolones (**4, 5**), from *Ormocarpum kirkii* was explained as result of isomerization in aqueous solution [32]. The same observation was reported in three isolated isoflavanones from *Platycelphium voënse* and *Desmodium canum* [41, 47]. Due to the positive cotton effect at 337 nm, the (3*R*) orientation was assigned to eryzerin B (**6**). However, eryzerin A (**7**) was reported in the same publication with undetermined absolute stereochemistry [44]. The (3*R*) orientation was also assigned to 2,3-dihydro-7-demethylrobustigenin (**8**) and saclenone (**9**) isolated from *Erythrina sacleuxii* based on the positive cotton effect at 320 and 334 nm, respectively [49].

Isoflavans configuration is much more complicated. The heterocyclic ring C is expected to have the half-chair form a fact that can be diagnosed from the vicinal coupling constants between H-2, H-3 and H-4 protons. Such *J* values along with the CD curves can then lead to determination of the absolute configuration [64]. (3*S*)-isoflavans with oxygenation at both the A and B rings display positive and negative cotton effects at 240 and 270–280 nm regions, respectively. The opposite was observed for the (3*R*)-enantiomers. The 7-deoxy (3*S*)-isoflavans

band II [39, 47, 50]. However, more data are required to draw a solid conclusion.

235–245 and 320–337 nm along with a shoulder 287–300 nm [29, 30, 31, 35, 36].

/HCl is diagnostic for free 5-OH

induced 17–23 nm bathochromic shift in band II due

tion of free 7-hydroxyl group. The 10–14 nm shift with AlCl3

in ring A [19, 27, 28, 50, 60].

64 Flavonoids - From Biosynthesis to Human Health

the obtained results revealed that AlCl3

range reported for isoflavanones [63].

**3.3. Circular Dichroism (CD) Spectroscopy**

1 H- and 13C-NMR spectra provide key information for the identification of the isoflavonoids skeleton. The proton and carbon signals for positions 2–4 in ring C (**Table 1**) provide a unique feature for each class.

The simplest ring C spectrum is that of isoflavones as it shows only one downfield proton singlet for H-2. The oxygenated C-2 chemical shift is also characteristic for isoflavones. The wide range for C-4 carbonyl resulted from the effect of C-5 substitutions. The lack of C5 free hydroxyl resulted in the upfield shift of the C-4 carbonyl chemical shift to a value less than 175.0 ppm in most cases [27, 34]. With the presence of C-5 free hydroxyl and formation of hydrogen bond C-4 carbonyl, the carbonyl chemical shift value is usually above 180.0 ppm [19, 24, 28].

Saturation of the double bond between C-2 and C-3 of isoflavones leads to the formation of the isoflavanone skeleton. Such array contains a CH2 -O and CH-aryl and renders the


**Table 1.** Key 1 H- and 13C-NMR spectral data for identification of isoflavonoid classes.

1 H-NMR signals of ring C more complex making an AMX spin system. The three protons appear as dd with different *J* values due to *ax-ax, ax-eq* and/or *eq-eq* splitting. In some cases, some signals may appear as *t* or interfere with other signals in the molecule [23, 41, 55, 61, 62]. Absolute configuration of isoflavanones was determined by a simple 1 H-NMR experiment in the presence of (*R*)- and (*S*)-binol as chiral solvating agent. The presence of (*R*)- or (*S*)-binol produces variable changes in the chemical shifts of the most downfield H-2 proton. Comparing these chemical shift changes enables the assignment of the absolute configuration [66].

No significant difference can be observed when the chemical shifts of positions 2–4 are compared in the 1 H-and 13C-NMR spectra of isoflavanones and homoisoflavonoids. The splitting pattern of H-3 is expected to be much more complex. However, the additional C-9 in homoisoflavonoids provides the key evidence for their identification. The H-9 protons appear in the range of δH 2.62–3.13 (dd) as a result of coupling with H-3 proton. The C-9 methylene appears at δC 31.9–32.2 ppm [63, 67].

Isoflavans lacks the C-4 carbonyl present in isoflavanones with expected two more proton signals from ring C to form an ABMXZ spin system. Although the H-4 proton signals are more upfield compared to H-2 and H-3, the splitting pattern is more complex than the corresponding isoflavanones. This pattern along with the 13C-NMR chemical shifts of C-2, C-3 and C-4 is the diagnostic feature for the isoflavan nucleus [20–22]. Isoflavan-4-ol is characterized by two oxygenated methines in both 1 H- and 13C-NMR spectra.

Formation of double bond between C-3 and C-4 in isoflavans led to the emerging of the isoflav-3-ene class. The ring C 1 H-NMR signals of isoflavenes is simplified to two singlet for the 2H of C-2 and 1H of C-4. In some reports, a long-range coupling with small *J* value (1–2 Hz) was observed between H-2 and H-4 protons [35, 36, 43, 56].

#### *3.4.2. 2D-NMR*

1 H-NMR and different 13C-NMR experiments like Distortionless Enhancement by Polarization Transfer (DEPT 45, DEPT 90 and DEPT 135) in most cases enable the identification of the main skeleton of the isoflavonoids as well as the substitution pattern. Heteronuclear Single-Quantum Correlation (HSQC) experiment is applied to correlate protons and carbons through one bond. So, assignment of protons and carbons as CH3 , CH2 and CH can be confirmed undoubtfully. 1 H-1 H-Correlation Spectroscopy (COSY) or similar experiments are applied to identify the spin systems in the compounds. These experiments identified protons separated by 3 bonds as well as different arrays present in the aromatic systems. The obtained COSY data allow the identification of the adjacent groups in the compounds and substitution pattern in the aromatic systems. Heteronuclear Multiple-Bond Correlation (HMBC) experiment acquired at different *J* values can identify correlation between protons and carbons through 2, 3 or sometimes 4 bonds especially in the aromatic systems. HMBC data play a key role in the determination of substituents location on the main skeleton. For example, the location of the furan ring in 4′-O-methylerythrinin C (**20**) at C-6 was assigned from HMBC correlations [28]. The location of the prenyl group at C-8 in erysubin F (**21**) was also assigned from correlations obtained from HMBC experiment [42].

Nuclear Overhauser Effect (NOE) is an effect observed between protons close to each other in space regardless to the number of bonds separating them [68]. The NOE effect can be clarified via One dimensional Nuclear Overhauser effect (1D-NOESY), Gradient-Enhanced Nuclear Overhauser Effect (GOESY) experiments or the now more favorable 2D-NOESY or Rotating Frame Nuclear Overhauser Effect (ROESY) experiments. The NOE effect is sometimes crucial for correct assignments of substitutions especially in the absence of significant UV data with shift reagents that can give information about OH group positions. The NOE effect in some situations is more decisive than HMBC due to the few number of correlations that can be observed and the fact that correlations are dependent on distance in space rather than direct bond correlations.

The positions of ring B substituents in lysisteisoflavanone (**22**) were assigned utilizing GOESY experiment where irradiation of the OCH3 and H-1″ of the prenyl group resulted in enhancement in their neighboring protons [50]. The NOE enhancement experiment was utilized to determine the position of OCH3 in olibergin B (**23**) [24]. Position of OCH3 in platyisoflavanone B (**24**) [41], vestitol (**25**), lotisoflavan (**26**) [21], erypoegin D (**27**) [43] and eryzerin B (**6**) [44] was assigned based on NOESY experiment results. The NOESY experiment was also employed to determine the position of glucose in ormosinoside A (**28**) [25].

NOESY data were also utilized to analyse the relative stereochemistry of the isoflavanol pumilanol (**29**) ring C protons [46].

#### **3.5. Mass Spectroscopy (MS)**

1

**Table 1.** Key 1

lute configuration [66].

**1**

66 Flavonoids - From Biosynthesis to Human Health

4.34–4.63 (dd, eq)

**Isoflavan-4-ol** 4.21–3.60 (dd, t) 66.8–66.9 3.52–3.49

tdd, dt, dd)

**Isoflavanones** 4.46–4.76 (dd, ax)

**Isoflavans** 4.33–3.83 (t, ddd,

**H 13C <sup>1</sup>**

at δC 31.9–32.2 ppm [63, 67].

oxygenated methines in both 1

flav-3-ene class. The ring C 1

*3.4.2. 2D-NMR*

1

pared in the 1

H-NMR signals of ring C more complex making an AMX spin system. The three protons appear as dd with different *J* values due to *ax-ax, ax-eq* and/or *eq-eq* splitting. In some cases, some signals may appear as *t* or interfere with other signals in the molecule [23, 41,

**Position 2 Position 3 Position 4**

**Isoflavones** 7.82–8.45 s 150.9–155.0 – 121.5–125.5 – 173.9–181.5

**Homoisoflavonoids** 4.06–4.32 (dd) 68.8–69.3 2.65–2.80 (m) 46.8–48.7 – 192.7–198.3

69.2–71.2 3.36–3.55 (tdd,

(ddd)

**Isoflavenes** 4.83–5.25 (s, d) 67.6–68.8 – 127.5–129.6 6.47–6.74 (s, d) 118.3–121.9

dd, dddd, m)

**H 13C <sup>1</sup>**

69.6–72.3 3.93–4.32 (dd) 45.3–51.1 – 193.0–198.8

30.79–33.6 2.64–2.98 (dd, ddd)

40.5–40.6 5.47–5.49 (d) 79.0–79.6

**H 13C**

experiment in the presence of (*R*)- and (*S*)-binol as chiral solvating agent. The presence of (*R*)- or (*S*)-binol produces variable changes in the chemical shifts of the most downfield H-2 proton. Comparing these chemical shift changes enables the assignment of the abso-

No significant difference can be observed when the chemical shifts of positions 2–4 are com-

pattern of H-3 is expected to be much more complex. However, the additional C-9 in homoisoflavonoids provides the key evidence for their identification. The H-9 protons appear in the range of δH 2.62–3.13 (dd) as a result of coupling with H-3 proton. The C-9 methylene appears

Isoflavans lacks the C-4 carbonyl present in isoflavanones with expected two more proton signals from ring C to form an ABMXZ spin system. Although the H-4 proton signals are more upfield compared to H-2 and H-3, the splitting pattern is more complex than the corresponding isoflavanones. This pattern along with the 13C-NMR chemical shifts of C-2, C-3 and C-4 is the diagnostic feature for the isoflavan nucleus [20–22]. Isoflavan-4-ol is characterized by two

Formation of double bond between C-3 and C-4 in isoflavans led to the emerging of the iso-

2H of C-2 and 1H of C-4. In some reports, a long-range coupling with small *J* value (1–2 Hz)

H-NMR and different 13C-NMR experiments like Distortionless Enhancement by Polarization Transfer (DEPT 45, DEPT 90 and DEPT 135) in most cases enable the identification of the

H-NMR signals of isoflavenes is simplified to two singlet for the

H- and 13C-NMR spectra.

was observed between H-2 and H-4 protons [35, 36, 43, 56].

H-and 13C-NMR spectra of isoflavanones and homoisoflavonoids. The splitting

H-NMR

26.1–31.9

55, 61, 62]. Absolute configuration of isoflavanones was determined by a simple 1

H- and 13C-NMR spectral data for identification of isoflavonoid classes.

Mass spectroscopy with different techniques and the great advances in instrumentation can provide accurately the molecular weight and the exact molecular formula. In addition, some common routes of fragmentation can provide additional evidences about the substitution pattern on both rings A and B. The mass fragments derived from a *retro*-Diels Alder (RDA) type cleavage give an idea about the substituent's on ring A and ring B as well (**Figure 2**). These MS fragments were used for the confirmation of ring A and ring B substitution pattern in the structure elucidation. Observation of MS ion fragments at *m/z* 177 and 153 as a result of *RDA* type cleavage followed by a hydrogen transfer indicated the location of two

**Figure 2.** Main fragments of retro-diels–alder (RDA) type cleavage.

methoxyls and a hydroxyl group on the B ring of the isoflavone olibergin A (**30**) [24]. The placement of two hydroxyl group at ring A and methylenedioxy and one methoxyl at ring B in the structure of (±)5,7-dihydroxy-2′-methoxy-3′,4′-methylenedioxyisoflavanone (**31**) was confirmed by MS fragments [33]. (S)-Platyisoflavanone A (**32**) mass spectrum showed fragment at *m/z* 232 indicating two methoxyls and 3-methylbut-2-enyl group at ring B [41]. The base peak in the MS spectrum of uncinanone D (**33**) at *m/z* 194 [C11H14O<sup>3</sup> ] resulted from *retro*-Diels Alder (RDA) cleavage of ring C supported the presence of 3 methoxyl groups at ring B [48]. Similarly, the location of three methoxyl groups on ring B and two hydroxyl groups on ring A in the structure of the isoflavanone (±)5,7-Dihydroxy-2′,3′,4′-trimethoxy-isoflavanone (**34**) was supported by MS fragmentation [33]. The fragmentation of 5,7-Dihydroxy-2′,4′,5′ trimethoxyisoflavanone (**35**) generated mass fragments at *m/z* 153 corresponding with ring A with two hydroxyls and at *m/z* 194 for ring B with three methoxyls [39]. The location of the methyl group in desmodianone A (**36**), desmodianone B (**37**), desmodianone D (**38**), desmodianone E (**39**) and 6-methyltetrapterol A (**40**) at C-6 was confirmed from the MS fragment at *m/z* 167 for A-ring [47]. The MS fragments at *m*/*z* 346 [508−163+H]+ and 194 indicated the presence of a sugar moiety in the A ring and three methoxyl groups in the B ring in the structure of 5,7-dihydroxy-2′,3′,4′-trimethoxy-isoflavanone 7-O-β-glucopyranoside (**41**) [33]. With a fragment 30 mass units less at *m/z* 164 in the spectrum of 5,7-Dihydroxy-2′,4′-dimethoxyisoflavanone 7-O-β-glucopyranoside (**42**), only two methoxyls were assigned to ring B and sugar was placed on ring A [33].

In addition to providing the M+ at 328 *m/z* of 2-methoxyjudaicin (**43**) the fragment at *m/z* 297 due to loss of the two methoxyls was very supportive for the structure since the MS spectrum of judaicin (**44**) show only fragment due to loss of one methoxyl group at C-2'. The MS data of judaicin 7-*O*-glucoside (**45**) and judaicin 7-*O*-(6″-*O*-malonylglucoside) (**46**) showed common ion at *m/z* 298 corresponding to the aglycone part after the loss of the glycosyl moieties at C-7 [30, 31].

#### **4. Isolated compounds update**

The isolated isoflavonoids from natural sources are presented in **Tables 2**–**6**, and their structures are provided in **Figures 3**–**7**. Isoflavones, isoflavanones and isoflavans from 2000 to date are arranged according to publication date in **Tables 2**–**4**, respectively. Due to the limited number of isoflavenes, the current survey includes all isolated members available in the literature (**Table 5**). Synthetic compounds are not included in this chapter.


Cajanin (**90**), Lachnoisoflavone A (**91**)

methoxyls and a hydroxyl group on the B ring of the isoflavone olibergin A (**30**) [24]. The placement of two hydroxyl group at ring A and methylenedioxy and one methoxyl at ring B in the structure of (±)5,7-dihydroxy-2′-methoxy-3′,4′-methylenedioxyisoflavanone (**31**) was confirmed by MS fragments [33]. (S)-Platyisoflavanone A (**32**) mass spectrum showed fragment at *m/z* 232 indicating two methoxyls and 3-methylbut-2-enyl group at ring B [41]. The

Diels Alder (RDA) cleavage of ring C supported the presence of 3 methoxyl groups at ring B [48]. Similarly, the location of three methoxyl groups on ring B and two hydroxyl groups on ring A in the structure of the isoflavanone (±)5,7-Dihydroxy-2′,3′,4′-trimethoxy-isoflavanone (**34**) was supported by MS fragmentation [33]. The fragmentation of 5,7-Dihydroxy-2′,4′,5′ trimethoxyisoflavanone (**35**) generated mass fragments at *m/z* 153 corresponding with ring A with two hydroxyls and at *m/z* 194 for ring B with three methoxyls [39]. The location of the methyl group in desmodianone A (**36**), desmodianone B (**37**), desmodianone D (**38**), desmodianone E (**39**) and 6-methyltetrapterol A (**40**) at C-6 was confirmed from the MS fragment

presence of a sugar moiety in the A ring and three methoxyl groups in the B ring in the structure of 5,7-dihydroxy-2′,3′,4′-trimethoxy-isoflavanone 7-O-β-glucopyranoside (**41**) [33]. With a fragment 30 mass units less at *m/z* 164 in the spectrum of 5,7-Dihydroxy-2′,4′-dimethoxyisoflavanone 7-O-β-glucopyranoside (**42**), only two methoxyls were assigned to ring B and

In addition to providing the M+ at 328 *m/z* of 2-methoxyjudaicin (**43**) the fragment at *m/z* 297 due to loss of the two methoxyls was very supportive for the structure since the MS spectrum of judaicin (**44**) show only fragment due to loss of one methoxyl group at C-2'. The MS data of judaicin 7-*O*-glucoside (**45**) and judaicin 7-*O*-(6″-*O*-malonylglucoside) (**46**) showed common ion at *m/z* 298 corresponding to the aglycone part after the loss of the glycosyl moieties

The isolated isoflavonoids from natural sources are presented in **Tables 2**–**6**, and their structures are provided in **Figures 3**–**7**. Isoflavones, isoflavanones and isoflavans from 2000 to date are arranged according to publication date in **Tables 2**–**4**, respectively. Due to the limited number of isoflavenes, the current survey includes all isolated members available in the lit-

erature (**Table 5**). Synthetic compounds are not included in this chapter.

] resulted from *retro*-

and 194 indicated the

base peak in the MS spectrum of uncinanone D (**33**) at *m/z* 194 [C11H14O<sup>3</sup>

**Figure 2.** Main fragments of retro-diels–alder (RDA) type cleavage.

68 Flavonoids - From Biosynthesis to Human Health

at *m/z* 167 for A-ring [47]. The MS fragments at *m*/*z* 346 [508−163+H]+

sugar was placed on ring A [33].

**4. Isolated compounds update**

at C-7 [30, 31].


**Table 2.** Isolated isoflavones from natural sources since 2000 to date.


**Name Source Ref.**

5,6-Dihydroxy-7,8,3′,5′-tetramethoxyisoflavone (**109**) *Iris pseudacorus* [79]

Formononetin (**58**) *Dalbergia oliveri* [53]

Neoraudiol (**111**) *Neorautanenia mitis* [52] Genistin (**1**), Daidzein (**64**), Daidzin (**65**), Puerarin (**112**) *Pueraria lobata* [34]

Isosideroxylin (**117**) *Leiophyllum buxifolium* [84] Achyranthoside A (**118**), Achyranthoside B (**119**) *Achyranthes bidentata* [6] Genistein (**57**), Biochanin A (**59**), Prunetin (**88**), Tectorigenin (**120**) *Dalbergia odorifera* [85]

*Antheroporum pierrei* [77]

*Potentilla astracanica* [7]

*Erythrina addisoniae* [23]

*Ormosia henryi* [25]

*Trifolium scabrum* [78]

*Astragalus mongholicus* [80]

*Dalbergia odorifera* [81]

*Ononis angustissima* [82]

*Piscidia carthagenensis* [83]

*Iris germanica* [26]

[40]

[9]

*Erythrina senegalensis*

*CMU-99*

Pierreione A (**92**), Pierreione B (**93**), Pierreione C (**94**), Pierreione

Genistein 5-*O*-*β*-glucopyranoside (**96)**, Prunetin 5-*O*-*β*-

70 Flavonoids - From Biosynthesis to Human Health

Erysubin F (**21**), Erythraddison I (**98**), Erythraddison II (**99**)

Daidzin (**65**), Sissotrin (**83**), 7-O-Methylbiochanin A (**63**)

Sophoricoside (**104**), Isoprunetin-7-O-*β*-D-glucoside (**105**)

Genistein (**57**),Biochanin A (**59**), Daidzein (**64**) 3′-Hydroxydaidzein-7-O-glucopyranoside (**107**) Calycosin-7-O-glucopyranoside (**108**)

Formononetin (**58**), Ononin (**82**), Calycosin (**81**) Calycosin-7-O-glucopyranoside (**108**)

Ormosinosides A (**28**), Genistein (**57**), Biochanin A (**59**), Daidzein

Isoformononetin (**101**), 4′,7-Di-O-methyldaidzein (**102**), Isoprunetin

Genistein (**57**), Biochanin A (**59**), Calycosin-7-O-glucopyranoside

Formononetin (**58**), Ononin (**82**), 3-(4-(Glucopyranosyloxy)-5 hydroxy-2-methoxyphenyl)-7-hydroxy-4H-chromen-4-one (**113**)

8-Hydroxyirilone 5-methyl ether (**121**), 8-Hydroxyirilone (**122**) Irilone 4′-methyl ether (**123**), Irilone (**124**), Irisolidone (**125**) Irigenin S (**126**), Irigenin (**127**), Iridin S (**128**), Iridin (**129**)

**Table 2.** Isolated isoflavones from natural sources since 2000 to date.

4′-*O*-*β*-d-glucopyranoside (**130**)

7,2′,5′-Trimethoxy-3′,4′-methylenedioxyisoflavone (**114**) 6,7-Dimethoxy-3′,4′-methylenedioxyisoflavone (**115**) 5,4′-Dihydroxy-7,2′,5′-trimethoxyisoflavone (**116**)

Neobavaisoflavone (**110**) *Erythrina excels*,

Biochanin A (**59**) *Dothideomycetes fungus*

D(**95**)

(**64**)

(**103**)

(**108**)

glucopyranoside (**97**)

Echrenone b10 (**100**)

6″-*β*-D-Xylose-genistin (**106**)



**Table 3.** Isolated isoflavonones from natural sources since 2000 to date.

**Name Source Ref.**

Desmodianone F (**171**), Desmodianone G (**172**) *Desmodium canum* [93]

Glabraisoflavanone A (**176**), Glabraisoflavanone B **(177)** *Glycyrrhiza glabra* [95] Isodarparvinol B (**178**), Dalparvin (**179**), (3*S*)-Sativanone (**180**) *Dalbergia parviflora* [96] 2′,2,5-Trimethoxy-6,7-methylenedioxyisoflavanone (**181**) *Iresine herbstii* [5] Erythraddison III (**182**), Erythraddison IV (**183**) *Erythrina addisoniae* [23] Dalbergioidin (**153**) *Lespedeza cyrtobotrya* [29]

Triquetrumone E (**186**), Triquetrumone F (**187**) *Tadehagi triquetrum* [98] Hirtellanine H (**188**), Hirtellanine I (**189**), Hirtellanine J (**190**) *Campylotropis hirtella* [99] Ormosinol (**191**) *Ormosia henryi* [25]

(+)-Violanone (**195**) *Dalbergia oliveri* [53]

*Echinosophora koreensis* [92]

*Erythrina costaricensis* [94]

*Campylotropis hirtella* [97]

*Ormocarpum kirkii* [32]

*Platycelphium voënse* [41]

*Dalbergia odorifera* [100]

*Uraria clarkei* [101]

*Cassia siamea* [102]

*Campylotropis hirtella* [103]

[40]

*Erythrina senegalensis*

3-Hydroxy-kenusanone B (**168**), Sophoraisoflavanone A (**169**)

72 Flavonoids - From Biosynthesis to Human Health

5,7,3′-Trihydroxy-4′-methoxy-6,5′-di(γ, γ-dimethylallyl)-isoflavanone

5,3′-Dihydroxy-4′-methoxy-5′-γ,γ-dimethylallyl-2″,2″-dimethylpyrano[5,

5,3′-Dihydroxy-2″,2″-dimethylpyrano[5, 6: 6,7]-2′″,2′″-dimethylpyrano[5,

3(*R*)-2′-Methoxyl-5,7,4′-trihydroxy-6-(3-methylbut-2-enyl)-isoflavanone

7-O-Glucosyldiphysolone (**2, 3**), (3*R*)-7,4′-Di-O-glucosyldiphysolone (**4**) (3*S*)-7,4′-Di-O-glucosyldiphysolone (**5**), 4″-hydroxydiphysolone (**192**)

3′-Geranyl-3,5,7,2′,4′-pentahydroxyflavonol (**185**)

Platyisoflavanone B (**24**), Platyisoflavanone A) (**32**) Platyisoflavanone C (**193**), Platyisoflavanone D (**113**) Sophoraisoflavanone A (**169**), Glyasperin F (**194**)

(3*S*)-2′,4′-Dimethoxy-3,7-dihydroxyisoflavanone (**196**) (3*S*)-2′,4′,5′-Trimethoxy-7-hydroxyisoflavanone (**197**) (3*R*)-4′-Methoxy-2′,3,7-trihydroxyisoflavanone (**198**) (3*R*)-Violanone (**199**), (3R)-3′-O-methylviolanone (**200**)

(3*R*) 5,7,3′,4′-Tetrahydroxy-2′-methoxyisoflavanone (**202**) (3*R*) 5′,8-Di-(γ,γ-dimethylallyl)-2′,5-dihydroxyl-4′,7-dimethoxyl-

3(R)-6,3′-di(3-hydroxy-3-methylbutyl)-2′-methoxyl-5,7,4′-

5,7-dihydroxy-2′-methoxy-3′,4′-methylenedioxy isoavanone (**155**) (3*R*) 7,2′,4′-Trihydroxy-3′-methoxy-5-methoxycarbonylisoflavanone (**205**) (3*R*) 7,2′-Dihydroxy-3′,4′-dimethoxy-5-methoxycarbonylisoflavanone

6,3′-di(3-hydroxy-3-methylbutyl)-5,7,2′, 4′-tetrahydroxyisoflavanone (**208**)

Sigmoidin H (**207**) *Erythrina excels*,

5,7-Dihydroxy-2′,4′-dimethoxyisoflavanone (**204**)

(3*R*)-Sativanone (**201**)

Dalbergioidin (**153**)

isoflavanone (**203**)

Uncinanone E (**150**)

trihydroxyisoflavanone (**209**)

(**206**)

Kenusanone H (**170**)

6: 6,7]isoflavanone (**174**)

6: 5,4]isoflavanone (**175**)

(**173**)

(**184**)


**Table 4.** Isolated homoisoflavonoids from natural sources since 2000 to date.



**Table 5.** Isolated isoflavans from natural sources since 2000 to date.


**Table 6.** Isolated isoflavenes from natural sources.

**Name Source Ref.**

Kotstrigoisoflavanol (**242**) *Kotschya strigosa* [110]

**Name Source Ref.** Neorauflavene (**243**) *Neorautanenia edulis* [51] Sepiol (**244**), 2′-O-Methylsepiol (**245**) *Gliricidia speium* [111] Dimethoxytrihydroxyisoflavene (**246**) *Baphia nitida* [56] Haginin A (**247**), Haginin B (**248**) *Lespedeza cyrtobotrya* [35]

2-Methoxyjudaicin (**43**) *Cicer bijugum* [30]

Haginin C (**251**), Haginin D (**252**) *Lespedeza cyrtobotrya* [113] Haginin D (**253**), Haginin E (Phenoxodiol) (**254**) *Lespedeza homoloba* [36] Erypoegin A (**255**), Erypoegin B (**256**) *Erythrina poeppigiana* [43] Glabrene (**257**) *Glycyrrhiza glabra* [114] Haginin A (**247**) *Lespedeza cyrtobotrya* [29] Haginin E (Phenoxodiol) (**254**) *Dothideomycetes* fungus CMU-99 [9]

*Erythrina livingstoniana* [37]

*Millettia* sp. [112]

*Cicer judaicum* [31]

Erylivingstone J (**10**), Erylivingstone K (**11**) 2′-Methoxyphaseollinisoflavan (**12**) 7, 4′-Dihydroxy-2′,5′-dimethoxy isoflavan (**13**)

7,3′,4′-Triacetoxy-6′-methoxyisoflav-3-ene (**249**) 7, 2′-Diacetoxy-4′-methoxyisoflav-3-ene (**250**)

**Table 6.** Isolated isoflavenes from natural sources.

Judaicin (**44**), Judaicin 7-*O*-glucoside (**45**) Judaicin 7-*O*-(6″-*O*-malonylglucoside) (**46**)

7,4′-Dihydroxy-2′-methoxy-3′-(3-methylbut-2-enyl) isoflavan (**14**)

**Table 5.** Isolated isoflavans from natural sources since 2000 to date.


**Figure 3.** Isolated isoflavones from natural sources since 2000 to date.

**Figure 3.** Isolated isoflavones from natural sources since 2000 to date.



**Figure 4.** Isolated isoflavanones from natural sources since 2000 to date.

**Figure 5.** Isolated homoisoflavonoids from natural sources since 2000 to date.

**Figure 6.** Isolated isoflavans from natural sources since 2000 to date.

**Figure 4.** Isolated isoflavanones from natural sources since 2000 to date.

80 Flavonoids - From Biosynthesis to Human Health

**Figure 7.** Isolated isoflavenes from natural sources since 2000 to date.
