2. Structures and classification

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.

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.

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.

Figure 1. Basic structure of flavonoids.

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

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

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

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.

Generally, hydroxyl, methoxyl, methyl, isopentenyl, methylenedioxyl, benzyl, nitro groups

prospects as weak hormones at treating menopausal syndrome of women [14–17].

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

2. Structures and classification

18 Flavonoids - From Biosynthesis to Human Health

three rings of A/B/C as shown in Figure 1.

2.2. Various substituents at ring A and B

Figure 1. Basic structure of flavonoids.

and so on, could be substituted at ring A and/or B.

classified, as shown in Table 1.

Table 1. Main structure types of flavonoids.

#### 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 Cglycosides 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.

### 3. Extraction and isolation

#### 3.1. 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.

### 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

2.3. Glycosidation

Structure type

2.4. Formation of complexes

Table 1. Main structure types of flavonoids.

3. Extraction and isolation

coumarins and alkaloids.

3.1. Extraction

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

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-

Basic structure Structure type Basic structure

Isoflavanone Homoisoflavanone

20 Flavonoids - From Biosynthesis to Human Health

Rotenoid Xanthone

Pterocarpin Neoflavonoid

Complexes of flavonoids could be formed with other types of compounds, such as phenylpropanoids,

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

#### 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 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/(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

3.1.2.3. Microwave-assisted extraction

22 Flavonoids - From Biosynthesis to Human Health

3.1.2.5. Macroporous adsorption resin

chosen according to the nature of target constituents.

proteins during the preparation of soybean isoflavones [23].

been studied [24], and the best system has been determined.

3.1.2.7. Aqueous two phase extraction (ATPE)

3.1.2.4. Enzyme method

high temperature.

3.1.2.6. Ultrafiltration

3.2. Isolation

3.2.1. General methods

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].

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

It has been used in the separation and enrichment of flavonoids. Suitable types should be

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%

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/(NH4)2SO4 and acetone/K2HPO4 have

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 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.

#### 3.2.2. Application of new isolation technologies

### 3.2.2.1. 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.

#### 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 84C than at 25C. Also, the binding capacity for the most promising MIP and its corresponding NIP was higher at 84C. The binding capacity for the MIP was similar to 30 μmol/g at 25C and 120 μmol/g at 84C, while for the corresponding NIP, it was similar to 15 and 90 μmol/g, at 25 and 84C, respectively.
