Key 1H- and 13C-NMR spectral data for identification of isoflavonoid classes.
Isoflavonoids are interesting class of natural products due to their positive effects on human health. Isoflavonoids include isoflavones, isoflavanones, isoflavans, rotenoids and pterocarpans. Although they are reported from many plant families, most isoflavonoids are produced by the subfamily Papilionaceae of the Fabaceae. Various chromatographic methods have been applied for the purification of isoflavonoids. Simple Ultra Violet (UV) absorption spectra as well as both One and two dimensional NMR (1D- and 2D-NMR) are critical for the identification of isoflavonoids. Each class of isoflavonoids has its unique feature in both 1H- and 13C-NMR that enable their proper characterization. High Resolution Mass Spectrometry (HRMS) is a substantial tool in such challenge. In vitro experiments indicated that isoflavonoids possess antioxidant, antimutagenic, antiproliferative as well as cancer preventive effects. Epidemiological studies provide support for some of these effects on human. Members of this class also are reported to have antimicrobial activity. In this chapter, isoflavones, isoflavanones, isoflavans, homoisoflavonoids and isoflavenes will be discussed in relation to their occurrence, methods of purification, spectral characters helpful in structure elucidation as well as their biological importance.
Isoflavonoids are a large subclass of the most common plant polyphenols containing 15 carbon atoms known as flavonoids . In isoflavonoids (3-phenylchromans), the phenyl ring B is attached to heterocyclic ring C at position 3 rather than 2 in flavonoids . 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 . The biosynthesis of isoflavonoids, consequently, is considered as an offshoot from the flavonoids biosynthetic pathway . 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 represent the subclasses with additional ring D formation .
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 and biological activities will be covered starting from 2000 to date.
2. Extraction and purification
The most popular method used for extraction of isoflavonoids is maceration with either MeOH or EtOH containing various percentages of H2O at room temperature followed by liquid-liquid fractionation using solvents with different polarities [6, 10, 19–32]. Another method of extraction used MeOH or EtOH under reflux or in soxhlet apparatus [5, 33–36]. Mixture of MeOH and CHCl3 or CH2Cl2 (1:1) was also applied for extraction [37–41]. Other research groups extracted the plant materials with acetone [42–44], CHCl3 [45, 46], CH2Cl2 [47–50] or diethyl ether  at room temperature. Successive extraction starting with petroleum ether or hexane, CHCl3, EtOAc and MeOH using soxhelt apparatus [52–56] was also reported. The isoflavone contents of soybeans were extract using supercritical fluid extraction .
The majority of purification and isolation steps utilized silica gel in the form of column, Preparative Thin Layer Chromatography (PTLC) or Centrifugal Preparative Thin Layer Chromatography (CPTLC) [19, 21, 45]. Combination of silica gel and Sephadex LH-20 was also applied for isoflavonoid purification [6, 10, 54, 55]. In addition to silica gel, semi-preparative C18 High Performance Liquid Chromatography (HPLC) columns were used for final purification of isoflavonoids [23, 30, 31, 38, 48]. The polar
3. Spectroscopic identification
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 .
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 .
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
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
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
3.4. Nuclear Magnetic Resonance (NMR) Spectroscopy
3.4.1. 1H- and 13C-NMR
1H- 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.
|Position 2||Position 3||Position 4|
|4.46–4.76 (dd, ax)|
4.34–4.63 (dd, eq)
|4.06–4.32 (dd)||68.8–69.3||2.65–2.80 (m)||46.8–48.7||–||192.7–198.3|
|4.33–3.83 (t, ddd, tdd, dt, dd)||69.2–71.2||3.36–3.55 (tdd, dd, dddd, m)||30.79–33.6||2.64–2.98 (dd, ddd)||26.1–31.9|
|4.21–3.60 (dd, t)||66.8–66.9||3.52–3.49 (ddd)||40.5–40.6||5.47–5.49 (d)||79.0–79.6|
|4.83–5.25 (s, d)||67.6–68.8||–||127.5–129.6||6.47–6.74 (s, d)||118.3–121.9|
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 1H-NMR signals of ring C more complex making an AMX spin system. The three protons appear as dd with different
No significant difference can be observed when the chemical shifts of positions 2–4 are compared in the 1H-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 1H- 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 1H-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
1H-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. 1H-1H-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
Nuclear Overhauser Effect (NOE) is an effect observed between protons close to each other in space regardless to the number of bonds separating them . 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 (
NOESY data were also utilized to analyse the relative stereochemistry of the isoflavanol pumilanol (
3.5. Mass Spectroscopy (MS)
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
In addition to providing the M+ at 328
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.
|Erysubin F (|||
|Isoerysenegalensein E (|||
|Olibergin A (|
Biochanin A (
7-O-Methylbiochanin A (
|7-O-Geranylbiochanin A (|||
|Olibergin B (|||
|Biochanin A (|
|Erypoegin D (|
|Tlatlancuayin (2′,5-dimethoxy-6,7-methylenedioxyisoflavone) (|||
|Pierreione A (|||
|Erysubin F (|
Echrenone b10 (
|Ormosinosides A (|
|Biochanin A (|||
|Achyranthoside A (|||
|8-Hydroxyirilone 5-methyl ether (|
Irilone 4′-methyl ether (
Irigenin S (
|Bolusanthol B (|
|Erypoegin C (|||
|Eryzerin B (|||
|Erypoegin G (|||
2′,4′-Dihydroxy-6″-methyl-6″-(4‴-methylpent-3-enyl) pyrano(3″,2″:6,7)-isoflavanone (
|Desmodianone A (|
|Uncinanone A (|||
|Uncinanone D (|||
Dihydrobiochanin A (
Uncinanone A (
|5,3′-Dihydroxy-4′-methoxy-5′-(3-methyl-1,3-butadienyl)-2″,2″-dimethylpyrano[5, 6:6,7]isoflavanone (|
5,3′-Dihydroxy-5′-(3-hydroxy-3-methyl-1-butenyl)-4′-methoxy-2″,2″-dimethylpyrano[5, 6:6,7]isoflavanone (
|Sophoronol A (|
Sophoronol D (
|3-Hydroxy-kenusanone B (|
Kenusanone H (
|Desmodianone F (|||
|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, 6: 6,7]isoflavanone (
5,3′-Dihydroxy-2″,2″-dimethylpyrano[5, 6: 6,7]-2′″,2′″-dimethylpyrano[5, 6: 5,4]isoflavanone (
|Glabraisoflavanone A (|||
|Isodarparvinol B (|||
|Erythraddison III (|||
|Triquetrumone E (|||
|Hirtellanine H (|||
|Platyisoflavanone B (|
Platyisoflavanone C (
Sophoraisoflavanone A (
|Uncinanone E (|
5,7-dihydroxy-2′-methoxy-3′,4′-methylenedioxy isoavanone (
|Sigmoidin H (|||
|6,3′-di(3-hydroxy-3-methylbutyl)-5,7,2′, 4′-tetrahydroxyisoflavanone (|
|Uncinanone D (|
Grabraisoflavanone A (
|Dothideomycetes fungus CMU-99|||
|Eryvarins Y (|
|Bolusanthol A (|||
|Eryzerin C (|||
|6-Desmethyldesmodian A (|
Desmodian B (
Desmodian C (
|Desmodian A (|||
|Vestitol (||Brazilian propolis|||
|Cordifoliflavanes A (|||
|Abruquinone A (|
Abruquinone K (
|Erylivingstone J (|
7, 4′-Dihydroxy-2′,5′-dimethoxy isoflavan (
7,4′-Dihydroxy-2′-methoxy-3′-(3-methylbut-2-enyl) isoflavan (
|Haginin A (|||
7, 2′-Diacetoxy-4′-methoxyisoflav-3-ene (
|Haginin C (|||
|Haginin D (|||
|Erypoegin A (|||
|Haginin A (|||
|Haginin E (Phenoxodiol) (|||
5. Biological activities
Isoflavonoids are reported to have a variety of bioprotective effects, including antioxidant, antimutagenic, anticarcinogenic and antiproliferative activities. Isoflavonoids may protect the body from hormone-related cancers, like breast, endometrial (uterine) and prostatic [115–119]. Isoflavonoids have gained a lot of public interest due to the possible correlation between their dietary consumption and health beneficial effects toward osteoporosis and post-menopausal symptoms [120, 121].
Among the isoflavonoids isolated from dothideomycetes fungus CMU-99, Biochanin A (
Haginin E (Phenoxodiol) (
Platyisoflavanone A (
Isoflavanones from the Stem of
As a part of plant phenolics, isoflavonoids are expected to have antioxidant activities. Ormosinol (
Other studies reported on the effects of isoflavonoids on specific enzymes are presented in Table 7.
|Achyranthoside A (|
Achyranthoside B (
|Lipopolysaccharide (LPS)-induced nitric oxide (NO) production||Significant inhibition|||
|Erysubin F (|
Echrenone b10 (
Erythraddison IV (
|Protein tyrosine phosphatase 1B (PTP1B)||Significant inhibition|||
|Sophoraisoflavanone A (|
Kenusanone H (
|Alcohol dehydrogenase (ADH)|
Aldehyde dehydrogenase (ALDH)
|Glabrene (||Tyrosinase inhibition||Significant inhibition|||
- Professor Dr. Ahmed A. Seif El-Dien, Department of Pharmacognosy, College of Pharmacy, Alexandria University, Alexandria 21215, Egypt. (Born in: 9 September 1948–passed away in: 19 December 2016).