Isolation and Structure Identification of Flavonoids

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 posi- tions 3, 5, 7, 3 0 , 4 0 and/or 5 0 . 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.


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][4][5][6][7][8][9]. The potential treatment and prevention effects have been shown in degenerative diseases such as tumors, aging and cardiovascular diseases [10][11][12][13][14][15]. Additionally, some compounds of flavonoids possess potential application prospects as weak hormones at treating menopausal syndrome of women [14][15][16][17].

Structures and classification
Flavonoids generally refer to the natural products of C 6 -C 3 -C 6 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.
According to connection mode of ring A with B, the connection position of ring B, the oxidation level of C 3 substructure and degree of polymerization, various type of flavonoids could be classified, as shown in Table 1.
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 C 6 -C 3 -C 6 ; few of them are C 6 -C 1 -C 6 (xanthone, for example). A few of them, such as homoisoflavones and rotenoids, possess C 6 -C 4 -C 6 structure skeleton. In most cases, C 3 part is formed to be a hexatomic or pentagon ring with C 6 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.

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.

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 Cglycosides is often connected to C-6 and/or C-8.

Formation of complexes
Complexes of flavonoids could be formed with other types of compounds, such as phenylpropanoids, coumarins and alkaloids.

Extraction
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.

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]. 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 CO 2 extraction of flavonoids from Licorice has been raised 2.2 times than the ordinary alcohol extraction [20].

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.

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].

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.

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.

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].

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 timesaving, 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/(NH 4 ) 2 SO 4 and acetone/K 2 HPO 4 have been studied [24], and the best system has been determined.

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.

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 C 18 silica gel) is commonly used to isolate flavonoid glycosides.

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.

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.

High-performance liquid chromatography (HPLC)
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.

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), C 18 , C 8 , C 2 , amino or phenyl columns could be applied, whereas C 18 and C 8 columns are mostly used among them.

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.

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.

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].

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.

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.

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

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 À1 region, carbonyl groups are in 1660-1680 cm À1 region and the vibrations of benzene rings are at about 1500, 1580 and 1600 cm À1 .

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 CDCl 3 , DMSO-d 6 , C 5 D 5 N, (CD 3 ) 2 CO and CD 3 OD, could be employed while performing NMR experiments. DMSO-d 6 is the optional solvent among them to perform NMR to flavonoids. Almost all kinds of flavonoids could be

4 0 -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].

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

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

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. Isoflavone, flavanone, flavanonol 3 0 ,4 0 -OH δ6.7-7.1 δ6.7-7.1 Table 6. Chemical shifts of protons on ring B of 3 0 ,4 0 -dioxygenated flavonoids.   Table 7. Chemical shifts of protons on ring B of various substituent modes [28].

Proton type Chemical shift
Phenolic Terminal protons of glycosyl δ4.5-5.5 Table 8. Chemical shifts of the protons on common substituents.

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 1 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 1 H and 13 C-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.  C-glycosides δ71-80 Table 13. Chemical shifts of carbons of common substituents on flavonoids.
As for the spectral method, the types and configurations could be speculated by the chemical shifts of glycosyl carbons in 13 C-NMR spectra, as shown in

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

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

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

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-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 Cotton effect (300-340) Absolute configuration

Flavans
The cotton effects of flavans are show in  Relative configuration between C-2 and C-3 Relative configuration between C-3 and C-4  The CE characteristics are shown in Table 23.
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 Cis-þ À 6aS, 11aS À þ 6aR, 11aR