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Flavonoids, which possess a basic C15 phenyl‐benzopyrone skeleton, refer to a series of compounds in which two benzene rings (ring A and B) are connected to each other through three carbon atoms. Based on their core structure, flavonoids can be grouped into different flavonoid classes, such as flavonols, flavones, flavanones, flavanonols, anthocyanidins, isoflavones and chalcones. Flavonoids are often hydroxylated in positions 3, 5, 7, 3′, 4′ and/or 5′. Frequently, one or more of these hydroxyl groups are methylated, acetylated, prenylated or sulfated. 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. This chapter mainly introduces the methods of isolation and structure identification of flavonoids.
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, China
Zhiyou Hao
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, China
Meng Li
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, China
*Address all correspondence to: fwsh@hactcm.edu.cn
1. Introduction
Flavonoids are important natural organic compounds of secondary metabolites that are produced during the long process of natural selection. They widely exist in the roots, stems, leaves, flowers and fruits of higher plants and ferns [1], which are of numerous categories and chemical structures. Because of the special chemical structures, flavonoids possess wide range of physiological and biochemical effects to the cells of mammal and other kinds of animals. Firstly, flavonoids possess strong chemical reactivity. For example, some flavonoids have antioxidant activity via scavenging free radicals in organism [2]. Furthermore, flavonoids possess various pharmacological activities of inhibiting the activity of enzymes, antitumor, antibiosis, antivirus, anti‐inflammatory and so on [3–9]. The potential treatment and prevention effects have been shown in degenerative diseases such as tumors, aging and cardiovascular diseases [10–15]. Additionally, some compounds of flavonoids possess potential application prospects as weak hormones at treating menopausal syndrome of women [14–17].
Flavonoids generally refer to the natural products of C6‐C3‐C6 basic structure. Most of them are the chromone derivatives with the core structure of 2‐phenylchromone and made up of three rings of A/B/C as shown in Figure 1.
Figure 1.
Basic structure of flavonoids.
According to connection mode of ring A with B, the connection position of ring B, the oxidation level of C3 substructure and degree of polymerization, various type of flavonoids could be classified, as shown in Table 1.
Table 1.
Main structure types of flavonoids.
The main factors of the structure diversity of flavonoids are as follows:
2.1. Change of ring system, degree of oxidation and number of core structure
Most of the flavonoids possess the core structure of C6‐C3‐C6; few of them are C6‐C1‐C6 (xanthone, for example). A few of them, such as homoisoflavones and rotenoids, possess C6‐C4‐C6 structure skeleton. In most cases, C3 part is formed to be a hexatomic or pentagon ring with C6 part. It could also be aliphatic chain, such as chalcone and dihydrochalcone. Supposing that the double bond of ring C was hydrogenated, dihydro derivative was formed, such as flavanone and flavononol. Commonly, ring B is connected to C‐2; it might be connected to C‐3 or C‐4 in a few cases, such as isoflavone and neoflavonoid. Most of the flavonoids have only one core structure; some of them possess two, however. We called them biflavonoids.
2.2. Various substituents at ring A and B
Generally, hydroxyl, methoxyl, methyl, isopentenyl, methylenedioxyl, benzyl, nitro groups and so on, could be substituted at ring A and/or B.
2.3. Glycosidation
Flavonoids are often glycosided to be O‐glycosides or C‐glycosides. During the glycosidation of flavonoids, almost every hydroxyl group could be the reaction position. However, the glycosidation reaction mostly occurred at 7‐OH of flavone, flavanone and isoflavone, 3‐ and/or 7‐OH of flavonol and flavanonol 3‐ and/or 5‐OH of anthocyanidin. The glycosyl group of C‐glycosides is often connected to C‐6 and/or C‐8.
2.4. Formation of complexes
Complexes of flavonoids could be formed with other types of compounds, such as phenylpropanoids, coumarins and alkaloids.
Traditional extraction methods of flavonoids often cause the problems of inefficiency, high energy consumption, more solvent consumption and so on. The new extraction methods and technologies occurred in recent years promoted the development of flavonoids. Because of the numerous types of flavonoids, the single extraction methods generally cannot meet the requirement. Traditional and modern methods should be applied together according to the extraction purpose.
3.1.1. Traditional extraction methods
Ethanol and methanol are frequently used to extract flavonoids. The common extraction methods include dipping, percolation, reflux, continuous reflux and so on. The alcohol of high concentration (90–95%) is applied to extract free flavonoids, and the alcohol at the concentration of about 60% is applied to extract flavonoid glycosides. For example, reflux method was applied to extract total flavonoids from leaves of Ginkgo biloba with 70% ethanol, and the product yield was significantly higher than the water decoction method [18].
Hot water extraction method is applied to flavonoid glycosides. It possesses the advantages of low cost, safety, simple equipment and could be applied in industrial production, but much water‐soluble impurities, such as proteins and saccharides might be mixed into the product.
Most of the flavonoids are acidic because of hydroxyl or carboxyl groups, so they could be extracted with alkaline water or alkaline dilute alcohol. The commonly used solvents include dilute sodium hydroxide, lime water, 5% sodium hydroxide dilute ethanol solution and so on. Water‐soluble impurities, such as tannins, pectins and mucilages, could be precipitated because of the formation of calcium salts during the extraction with lime water. It has often showed good results if 5% sodium hydroxide dilute ethanol solution was used. However, the product yield might be reduced because some flavonoids obtained after acidification might be dissolved in dilute ethanol solution. It should be noted that the concentration of alkali should not be excessive during the extraction, and the acidity of the solvent should not be excessive during acidification. Additionally, sodium borate should be used if adjacent phenolic hydroxyl groups are existed in the structures. Extraction of rutin from Flos Sophorae Immaturus is one example [19].
3.1.2. New extraction technologies
3.1.2.1. Supercritical fluid extraction (SFE)
The goal of selected extraction, isolation or purification might be achieved via controlling temperature, pressure and regulating the type and consumption of cosolvent during the supercritical fluid extraction. Cosolvent (e.g. ethanol) is usually added to induce product yield. For example, the product yield of supercritical CO2 extraction of flavonoids from Licorice has been raised 2.2 times than the ordinary alcohol extraction [20].
3.1.2.2. Ultrasonic extraction
This method has been used in the quality analysis and small amount extraction of flavonoids. It’s still seldom used in industrial production, however. For instance, ultrasonic extraction was used in the extraction of flavone from the bud of Sophora japonica, and the product yield was higher than reflux extraction method [21]. Ultrasonic extraction is superior to reflux method from the perspectives of energy saving, time saving and technology.
3.1.2.3. Microwave‐assisted extraction
It has obtained good results in the extraction of flavonoids. However, it is confined to laboratories so far. It also can be applied combined with other methods to induce product yield. For example, refluxing extraction was used after treatment with microwave for a short time during the extraction of flavonoids from Ophiopogon japonicus. The product yield was induced significantly [22].
3.1.2.4. Enzyme method
The impurities, such as starches, pectins and proteins, could be removed after enzymolysis. Long extraction time is the limitation of this method. However, the mild operational conditions could overcome the shortcomings that some bioactive components may be decomposed under high temperature.
3.1.2.5. Macroporous adsorption resin
It has been used in the separation and enrichment of flavonoids. Suitable types should be chosen according to the nature of target constituents.
3.1.2.6. Ultrafiltration
The molecules of different molecular weight are separated depend on the pressure difference between both sides of ultrafiltration membrane. Proteins, polypeptide, polymeric pigments and starches could be removed largely. It possesses advantages of simple operations such as no need to heat and destroy the molecular structures. It could remove 69.4% pectins and 66% proteins during the preparation of soybean isoflavones [23].
3.1.2.7. Aqueous two phase extraction (ATPE)
Aqueous two phase system (ATPS) is formed when either two polymers and kosmotropic salt, or two salts (one chaotropic salt and the other a kosmotropic salt) are mixed at appropriate concentrations and at a particular temperature. The distribution coefficients are different in specific ATPS of different substances. The separation objective will be achieved via selective distribution between the two phases after adding substances into the system. It possesses advantages of time‐saving, simple operation, mild condition, being easy to expand process, large treatment capacity and so on. The commonly used ATPS are high polymer system (e.g. PEG‐Dextran system), high polymer‐inorganic salt system and PEG‐sulfate/phosphate system. The distribution characteristics of puerarin in the two‐phase aqueous systems of PEG/(NH4)2SO4 and acetone/K2HPO4 have been studied [24], and the best system has been determined.
3.2. Isolation
3.2.1. General methods
The isolation of flavonoids includes the separation of flavonoids and other kinds of compounds, and the obtaining of monomeric compounds. The choosing of isolation methods is made primary according to polarity, acidity, molecular weight difference and special structure. Chromatography is still the first choice to isolate flavonoids.
3.2.1.1. Silica gel chromatography
Silica gel chromatography is the main method to isolate or identify flavonoids. It is applied to isolate low or medium polar constituents. Reversed phase silica gel (e.g. reversed phase C18 silica gel) is commonly used to isolate flavonoid glycosides.
3.2.1.2. Polyamide chromatography
Polyamide is a good adsorbent to isolate flavonoids. The adsorption strength hinges on hydrogen bonding associated between polyamide and flavonoids, which depends on the number and positions of hydroxyl groups in the molecules of flavonoids.
3.2.1.3. Polydextran gel chromatography
The most commonly used polydextran gel is sephadex LH‐20 during the isolation of flavonoids. Adsorption is the main mechanism during the isolation of free flavonoids, and the adsorption strength is mainly based on the phenolic hydroxyl groups. However, molecular sieve effect plays the leading roles during the isolation of flavonoid glycosides.
This technology has been widely used in the isolation and quality analysis of flavonoids and other kinds of natural products. The determination of chromatographic condition is the key to achieve separation purpose.
3.2.2.1.1. Choice of stationary phases
Silica gel and amino columns are mostly used during the operation of normal phase chromatography. In the reversed phase, HPLC (RP‐HPLC), C18, C8, C2, amino or phenyl columns could be applied, whereas C18 and C8 columns are mostly used among them.
3.2.2.1.2. Choice of mobile phases
Methanol‐water and acetonitrile‐water system are commonly applied in RP‐HPLC. In order to improve separation performance, minute quantity of acid (e.g. trifluoroacetic acid) could be added into mobile phase.
3.2.2.1.3. Detection
All of the flavonoids are able to absorb ultraviolet rays, so generally they could be detected by UV detectors. It is usually detected at 254–280 nm or 340–360 nm for flavones, flavonols and the corresponding glycosides, 520–540 nm for anthocyanidins and the corresponding glycosides, 250 nm for chromones.
3.2.2.2. High‐speed counter current chromatography (HSCCC)
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 (9.5:10.5) two‐phase system [25].
3.2.2.3. Molecular imprinting technology (MIT)
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 84°C than at 25°C. Also, the binding capacity for the most promising MIP and its corresponding NIP was higher at 84°C. The binding capacity for the MIP was similar to 30 μmol/g at 25°C and 120 μmol/g at 84°C, while for the corresponding NIP, it was similar to 15 and 90 μmol/g, at 25 and 84°C, respectively.
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].
Figure 2.
UV spectrum of flavonoids.
Structure type
Band II (nm)
Band I (nm)
Flavone
250–280
304–350
Flavonol (3‐OH is substituted)
250–280
328–357
Flavonol (3‐OH is free)
250–280
358–385
Isoflavone
245–270
310–330 (shoulder peak)
Flavanone and flavanonol
270–295
300–330 (shoulder peak)
Chalcone
220–270 (weak peak)
340–390
Aurone
230–270 (weak peak)
370–430
Anthocyanidin
270–280
465–560
Table 2.
The spectral characteristics of UV‐VIS spectrum of flavonoids.
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 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−1 region, carbonyl groups are in 1660–1680 cm−1 region and the vibrations of benzene rings are at about 1500, 1580 and 1600 cm−1.
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‐d6 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‐d6 as the solvent. The drawback of this solvent is high boiling point, which leads to difficulty in sample recovery.
4.3.1. 1H‐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 1H‐NMR spectrum.
4.3.1.1. Protons on ring C
1H‐NMR characteristics of protons on ring are shown in Table 3 [28].
Type
2‐H
3‐H
Note
Flavanone
δ6.3– 6.8 (s)
The signals maybe overlapped by H‐6 or H‐8.
Flavonol
None signal.
Isoflavone
δ7.6–7.8 (s)
The signal is at rather low field because of influence of oxygen atom at position 1 and carbonyl at position 4.
After glycosidation of 3‐OH, resonance signals of both H‐2 and H‐3 shift to low field.
Aurone
Exocyclic proton: δ6.5–6.7 (s)
Table 3.
Chemical shifts and coupling constants of ring C of common flavonoids.
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.
Figure 3.
Substitution modes of ring A.
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.
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, J=2.0, 8.0 Hz). H‐8 is showed to be a doublet (J=2.0Hz) 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].
Type
H‐5
H‐6
H‐8
5,7‐Dihydroxyl
Flavone, flavonol, isoflavone
δ6.0–6.2 d
δ6.3–6.5 d
7‐O‐Glucosides of above compounds
δ6.2–6.4 d
δ6.5–6.9 d
Flavanone, flavanonol
δ5.75–5.95 d
δ5.9–6.1 d
7‐O‐Glucosides of above compounds
δ5.9–6.1 d
δ6.1–6.4 d
7‐Hydroxyl flavonoids
Flavone, flavonol, isoflavone
δ7.9–8.2 d
δ6.7–7.1 d
δ6.7–7.0 d
Flavanone, flavanonol
δ7.7–7.9 d
5.7–6.0 d
δ5.9–6.1 d
5,6,7‐Trihydroxyl flavonoids
δ6.95 s
5,7,8‐Trihydroxyl flavonoids
δ6.3 s
Table 4.
Chemical shifts of protons on ring A.
4.3.1.3. Protons on ring B
There are a variety of substituted modes of ring B, such as non‐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 determined via the chemical shifts and coupling constants of ring B.
Figure 4.
Substitution modes of ring B.
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 meta‐coupling. The signals of H‐2’ and H‐6’ are usually at δ7.1–7.6 and of H‐3′, H‐4′ and H‐5′ are at δ7.9–8.2.
4.3.1.3.2. 4′‐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].
Type
H‐2′, H‐6′
H‐3′, H‐5′
Flavanone
δ7.1–7.3
δ6.5–7.1
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
Table 5.
Chemical shifts of protons on ring B of 4′‐oxygenated flavonoids.
4.3.1.3.3. 3′,4′‐Dioxygenation
In this circumstance, one ABX coupling system is formed by three aromatic protons, and three groups of signals are displayed as H‐2′(1H, d, J ≈ 2.0Hz), H‐5′(1H, d, J ≈ 8.0Hz) and H‐6′(1H, dd, J ≈ 2.0, 8.0Hz). The chemical shifts of protons on ring B are shown in Table 6[28].
Type
Substituent mode
H‐2′
H‐5′
H‐6′
Flavone
3′,4′‐OH and 3′‐OH, 4′‐OCH3
δ7.2–7.3
δ6.7–7.1
δ7.3–7.5
Flavonol
3′,4′‐OH and 3′‐OH, 4′‐OCH3
δ7.5–7.7
δ7.6–7.9
3′‐OCH3, 4′‐OH
δ7.6–7.8
δ7.4–7.6
3′,4′‐OH, 3‐O‐glc
δ7.2–7.5
δ7.3–7.7
Isoflavone, flavanone, flavanonol
3′,4′‐OH
δ6.7–7.1
δ6.7–7.1
Table 6.
Chemical shifts of protons on ring B of 3′,4′‐dioxygenated flavonoids.
4.3.1.3.4. 2′‐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′,4′,5′‐Trioxygenation
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′,4′,5′‐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′,4′‐Dioxygenation
In the cases of this mode, H‐3′ will be showed at δ6.00–6.6 (d, J ≈ 2.0 Hz), H‐5′ at δ 6.6–6.5 (dd, J ≈ 2.0, 8.0 Hz) and H‐6′ at δ7.0–7.4 (d, J ≈ 8.0 Hz). See Table 7.
Substituent mode
H‐2′
H‐3′
H‐4′
H‐5′
H‐6′
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)
2′‐Oxygenated
δ6.8–6.9 (m or dd)
δ7.2 (m or dd)
δ6.8–6.9 (m or dd)
7.4–7.5 (m or dd)
4′‐Oxygenated
δ7.1–8.1 (2H, d, J≈8.0Hz)
δ6.5–7.1 (2H, d, J≈8.0Hz)
δ6.5–7.1 (2H, d, J≈8.0Hz)
7.1–8.1 (2H, d, J≈8.0Hz)
2′,4′‐Dioxygenation
δ6.00–6.6(d, J≈2.0Hz)
δ6.30–6.50 (1H, dd, J≈8.0,2.0Hz)
7.0–7.4(d, J≈8.0Hz)
3′,4′‐Dioxygenation
δ7.2–7.8(1H, d, J≈2.0Hz)
δ6.7–7.1(1H, d, J≈8.0Hz)
6.7–7.9(1H, dd, J≈2.0, 8.0Hz)
3′,4′,5′‐Trioxygenation
δ6.5–7.5 (2H, s, H‐2′,6′) as identical oxygen‐bearing substituents are connected to position 3′,4′ and 5′.
2′,4′,5′‐Trioxygenation
δ6.4–6.6(s)
δ7.2–7.5(s)
Table 7.
Chemical shifts of protons on ring B of various substituent modes [28].
4.3.1.4. Common substituents
The proton chemical shifts of common substituents of flavonoids are shown in Table 8 [28].
Chemical shifts of the protons on common substituents.
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.
Type
C‐2
C‐3
C‐4
Flavone
δ160.0–165.5
δ104.0–112.0
δ175.0–184.0
Flavonol (3‐OH is free)
δ145.0–149.5
δ135.6–139.0
δ172.0–178.0
Flavonol (3‐OH is free)
δ155.7–157.0
δ133.2–134.3
δ172.0–178.0
Isoflavone
δ149.8–156.5
δ120.3–126.0
δ174.5–182.0
Flavanone
δ75.0–80.5
δ42.0–44.6
δ189.5–197.2
Flavanonol
δ82.7–83.5
δ71.2–73.0
δ188.0–197.0
Chalcone
δ136.9–145.4
δ116.8–128.1
δ188.0–194.6
Dihydrochalcone
δ39.2–45.5
δ28.5–31.2
δ199.6–205.5
Aurone
δ146.1–147.7
δ111.0–113.3
δ180.5–182.7
Flavan‐3‐ol
δ77.5–82.7
δ65.1–68.2
δ27.5–28.6
Flavan‐3,4‐diols
δ80.0–82.5
δ72.5–74.4
δ69.8–72.0
Table 9.
Chemical shifts of carbons in ring C of flavonoids [28].
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].
Ring A
Chemical shift
Ring B
Chemical shift
C‐5
δ124–126
C‐2′,6′
δ126.0–130.0
C‐6
δ124–126
C‐3′,5′
δ128–129
C‐7
δ133–135
C‐4′
δ128.5–132
C‐8
δ116.5–119
C‐1′ (flavone, flavonol, isoflavone)
δ130–133
C‐9
δ154–156
C‐1′ (flavanone, flavonol)
δ138–139
C‐10
δ121–124
Table 10.
Chemical shifts of carbons in ring A and B if they are not substituent.
Substituent mode
Type
C‐5
C‐6
C‐7
C‐8
C‐9
C‐10
5‐Oxygenated
≈δ160
δ109–111
δ135–137
δ107–108
δ157–160
δ111
7‐Oxygenated
Flavone, flavonol
δ126
δ114
δ162–164
δ100–102
δ157
δ114–116
Flavanone, flavanonol
δ128
δ109.5–110
δ165–166
δ100
δ162–164
δ114–116
Isoflavone
δ127
δ115
δ161–163
δ102–104
δ156–158
δ117–119
5,7‐Dioxygenation
Flavone, flavonol, isoflavone
δ160–162
δ97–100
δ162–165
δ93–95
δ155–158
δ103–109
Flavanone, flavanonol
δ162–163
δ93–97
δ165–167
δ93–97
δ161–163
δ100–104
Table 11.
Chemical shifts of carbons in ring A of flavonoids.
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].
Substituent mode
Type
C‐1′
C‐2′
C‐3′
C‐4′
C‐5′
C‐6′
4’‐Oxygenated
Flavone, flavonol, isoflavone
δ121–123
δ130
δ115
δ157–161
δ115
δ130
Flavanone, flavanonol
δ128–130
3′‐OH,4′‐OCH3 (or 3′,4′‐dihydroxyl)
Flavone, flavonol, isoflavone
δ121–125
δ113–114
δ145–147
δ149–151
δ112–116
δ118–122
3′‐OCH3,4′‐OH
δ121–125
δ110–111
δ150–152
δ148
δ115–117
δ118–122
3′‐OH,4′‐OCH3 (or 3′,4′‐dihydroxyl)
Flavanone, flavanonol
δ128–129
δ111–115
δ144–147
δ146–148
δ112–116
δ118–120
3′‐OCH3,4′‐OH
δ131–132
δ111–115
δ147–149
δ146–147
δ112–116
δ118–120
2’,4’‐Dioxygenation
Flavonoids
δ108–113
δ156–158
δ102–104
δ157–162
δ104–108
δ131–132
3′,4′,5′‐Trioxygenation
δ120–126
δ106–109
δ146–153
δ93–97
δ136–142
δ106–109
Table 12.
Chemicals shifts of carbons on ring B of flavonoids.
4.3.2.2.3. Signal characteristics of common substituents
The carbon chemical shifts of common substituents are shown in Table 13 [28].
Chemical shifts of carbons of common substituents on 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 1H‐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 1H 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].
Glycosylation position
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
7‐O‐glu
+0.8
−1.4
+1.1
+1.7
7‐O‐rha
+0.8
−2.4
+1.0
+1.7
3‐O‐glu
+9.2
−2.1
+1.5
+0.4
+1.0
−0.8
+1.1
−0.3
+0.7
+1.5
3‐O‐rha
+10.3
−1.1
+2.0
+0.6
+1.1
5‐O‐glu
−2.8
+2.2
−6.0
−2.7
+4.4
−3.0
+3.2
+1.4
+4.3
−1.3
−1.2
−0.4
−0.8
−1.0
−1.2
3′‐O‐glu
−0.5
+0.4
+1.6
+1.4
+0.4
+3.2
4′‐O‐glu
+0.1
+1.0
+3.7
+0.4
+2.0
−1.2
+1.4
Table 14.
Glycosylation shifts (average values) of flavonoids in 13C‐NMR spectrum.
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 1H‐NMR spectra. The absolute configurations, however, should be determined by chemical methods and gas chromatography.
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].
Type of glycosyl
C‐1′′
C‐2′′
C‐3′′
C‐4′′
C‐5′′
C‐6′′
β‐D‐glu
δ104.0
δ74.1
δ76.8
δ70.6
δ76.8
δ61.8
α‐D‐glu
δ100.0
δ72.2
δ74.1
δ70.6
δ72.5
δ61.6
β‐D‐gal
δ104.5
δ71.7
δ73.8
δ69.7
δ76.0
δ62.0
α‐ D‐ gal
δ100.1
δ69.2
δ70.5
δ70.2
δ71.6
δ62.2
β‐D‐man
δ102.3
δ71.7
δ74.5
δ68.4
δ77.6
δ62.6
α‐D‐man
δ102.2
δ71.4
δ72.1
δ68.3
δ73.9
δ62.5
β‐D‐rha
δ102.4
δ71.8
δ74.1
δ73.4
δ73.4
δ17.9
α‐D‐rha
δ102.1
δ71.2
δ71.5
δ73.3
δ69.5
δ17.9
Table 15.
Carbon chemical shifts of common glycosyls.
4.4. Mass spectral characteristics of 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 [M+H]+, [M+Na]+, [M+K]+ and [M+NH4]+ will be displayed. [2M+H]+, [2M+Na]+ 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.
Figure 5.
MS fragmentation pathway of flavone.
Figure 6.
MS fragmentation pathway of flavanone.
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.
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].
Type
Optical activity
Spatial structure
Flavan
–
2S
Flavanonol (trans‐form)
+
2R,3R
Epicatechin (cis‐form)
‐
2R,3R
Epicatechin (cis‐form)
+
2S,3S
Catechin (trans‐form)
+
2R,3S
Pterocarpin (cis‐form)
–
6aR, 11aR
6a‐Hydroxyl pterocarpin (cis‐form)
–
6aS, 11aS
6a‐Hydroxyl pterocarpin (trans‐form)
+
6aR, 11aR
Table 16.
Optical activities of flavonoids.
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.
CE
Absolute configuration of C‐2
270–290 nm
320–330 nm
−
+
S
+
−
R
Table 17.
Relationship between CE and absolute configurations of flavanones.
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 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].
Relative configuration
Cotton effect (300–340)
Absolute configuration
trans‐
+
2R, 3R
−
2S, 3S
cis‐
+
2R, 3S
−
2S, 3R
Table 18.
Relationship between absolute configurations of flavanonol with CE.
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].
Relative configuration
CE
Absolute configuration
240 nm
280 nm
Trans‐
+
−
2R, 3S
−
+
2R, 3R
Cis‐
−
+
2S, 3R
+
+
2S, 3S
Table 19.
Relationship between absolute configurations of 3‐hydroxyl flavans with CE.
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].
Relative configuration
CE
Absolute configuration
240 nm
280 nm
Trans‐
−
−
2S, 4S
+
+
2S, 4R
Cis‐
−
−
2S, 4S
+
+
2R, 4R
Table 20.
Relationship between absolute configurations of 4‐hydroxyl 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].
Relative configuration between C‐2 and C‐3
Relative configuration between C‐3 and C‐4
Cotton effect (280 nm)
Absolute configuration
Cis‐
Cis‐
+
2S,3S,4S
Trans‐
+
2S,3S,4R
Trans‐
Cis‐
+
2S,3R,4R
trans‐
+
2S,3R,4S
Cis‐
Cis‐
−
2R,3R,4R
Trans‐
−
2R,3R,4S
Trans‐
Cis‐
−
2R,3S,4S
Trans‐
−
2R,3S,4R
Table 21.
Relationship of absolute configurations of 3,4‐dihydroxyl flavans with CE.
4.5.2.6. Flavans
The cotton effects of flavans are show in Table 22 [34].
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 8.
Synthetic isoflavans (31a/b–36a/b).
Figure 9.
CD spectra of isoflavans with oxygenation at both the A and B rings.
4.5.2.8. Pterocarpins
The spectral characteristics are shown in Table 24 [36].
Relative configuration of between C‐6a and C‐11a
CE
Absolute configuration
260–310 nm
220–250 nm
Trans‐
+
6aS, 11aR
−
6aR, 11aS
Cis‐
+
−
6aR, 11aR
−
+
6aS, 11aS
Table 24.
Relationship of absolute configurations of pterocarpins with CE.
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].
Relative configuration of between C‐6a and C‐11a
CE
Absolute configuration
260–310 nm
220–250 nm
Trans‐
−
−
6aS, 11aR
+
+
6aR, 11aS
Cis‐
+
−
6aS, 11aS
−
+
6aR, 11aR
Table 25.
Relationship of absolute configurations of 6a‐hydroxyl pterocarpins with CE.
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Written By
Weisheng Feng, Zhiyou Hao and Meng Li
Submitted: 12 October 2016Reviewed: 10 February 2017Published: 23 August 2017
Open access peer-reviewed chapter
