**4. Derivation of flavonoids**

Due to the intrinsic health benefits possessed by flavonoids, numerous approaches have been developed during the past decades for the derivation of a wide range of flavonoids. Basically, these approaches can be divided into three major categories: traditional plant extraction, chemical synthesis, and biosynthesis.

### **4.1 Traditional plant extraction via organic solvents**

Traditionally, flavonoids are extracted from various plant species, which currently remains the most commonly used methods. During the past decades, researchers have developed plenty of methods to improve the yield and purity of flavonoids derived from plants. Generally, the plant tissues are air-dried and ground into powder for extraction via organic solvents (most commonly methanol and ethanol), and the extracts are then subjected to successive fractionation with other organic solvents (most commonly petroleum ether, chloroform, ethyl acetate, and n-butyl alcohol), followed by repeated silica gel and Sephadex LH-20 column chromatographies [44, 95]. The yield of plant-derived flavonoids can be improved by ultrasonic wave- [96], microwave- [97], and enzyme-assisted extraction [98]; aqueous two-phase extraction [99]; and a combination of these modifications [100]. The isolated flavonoids are then subjected to polyamide thin plate chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) analyses to determine their identity and purity [2, 3]. Due to the high solubility of most flavonoids in organic solvents, this strategy often demonstrates a high efficiency in the derivation of flavonoids from plant tissues. However, the disadvantage of the plant extraction is obvious. Due to the very low content of most flavonoids in plant tissues, the extraction and isolation of flavonoids often requires multiple

steps and plenty of time, labor, and organic solvents, which greatly increase the production cost. Moreover, different plant tissues often need to develop different approaches for processing, which makes the extraction more complicated and further increase the cost for the production of flavonoids. Therefore, this approach is not cost-effective, and it is crucial to develop alternative strategies to reduce the cost for producing flavonoids.

#### **4.2 Chemical synthesis of flavonoids**

Another approach for producing flavonoids is chemical synthesis. Basically, there are two strategies for chemical synthesis of flavones, that is, the chalcone route and the Baker-Venkataraman method [101]. Even though there are a few successful examples, chemical synthesis of flavonoids is often very complicated and involved in many steps [2]. It requires toxic reagents and extreme reaction conditions [3, 102]. Chiral synthesis and subsequent modifications further increase the difficulty of this approach in the production of flavonoids [3]. Moreover, the multistep chemical reactions often produce quite a number of intermediate products with a high similarity in structure, which further increases the difficulty in purification of the desired products. Therefore, chemical synthesis is not economically feasible for the mass production of flavonoids [3].

#### **4.3 Biosynthesis of flavonoids**

Since the biosynthetic pathway of flavonoids is largely elucidated in plants [20], other promising alternative strategies have been developed to produce these secondary compounds [2, 103–106]. One of these alternative strategies is to produce flavonoids in a microbial cell factory. It has been well known that *Escherichia coli* and *Saccharomyces cerevisiae* are the two most commonly used model organisms for the construction of a microbial cell factory. There are quite a few paradigms for the production of flavonoids using this strategy. For example, eriodictyol has been produced using l-tyrosine as a substrate in *E. coli* BL21(DE3) genetically modified by *TAL, 4CL, CHS, CHI, F3H*, and *F3'H* genes and the production can reach up to 107 mg/L by further introducing three other genes *acs*, *accBC*, and *dtsR1* to enhance the availability of malonyl-CoA [103]. Kaempferol has been produced in a microbial cell factory by introducing a *de novo* biosynthetic pathway into *S. cerevisiae*, and the biosynthesis has been further improved by introducing two more pathways to enhance the generation of acetyl-CoA and malonyl-CoA [107]. Obviously, this strategy circumvents some inherent disadvantages of traditional plant extraction and chemical synthesis. However, not all genetically modified microbes can produce desired products due to the well-known complexity of a microbial cell system, the incompatibility of artificially synthesized genetic elements in host cells, the growth inhibition of host cells by desired and intermediate products, and the instability of an engineered biosystem itself [2, 108].

Recently, we have developed an *in vitro* platform to produce flavonoids by constructing a multienzyme synthetic system to convert naringenin into kaempferol in one pot [2]. After optimizing a series of reaction parameters, including the components and pH value of the buffer system, reaction temperature and time, and total amount and ratio of the enzymes, the production yield can reach up to 37.55 ± 1.62 mg/L within 40–50 min with a conversion rate of 55.89% ± 2.74% [2]. The advantages of this strategy are obvious. It is time- and labor-saving. The reaction conditions are easy to control accurately. Due to the clearness in the buffer components and the lack of complex physiological regulation as occurred in the microbial cell factory, it is possible to easily make further optimization in the future.

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*Flavonoids and Pectins*

**5. Conclusions**

**Conflict of interest**

*DOI: http://dx.doi.org/10.5772/intechopen.84960*

It is also much easier to purify desired products from this *in vitro* synthetic system than from the cell factory because of the simplicity of the components in the system. In addition, the strategy is highly cost-effective because of the cheap chemicals and recombinant proteins used in this system. More importantly, the system is easy to scale up and therefore possesses a huge industrialization potential. It also provides a guide for other secondary metabolites to produce economically. However, problems still exist in this production strategy. For example, due to the lack of P450-reductase function, prokaryotically expressed cytochrome P450 enzymes lose their enzymatic activities [109]. To achieve a functional expression, Leonard and colleagues fused a plant P450 enzyme gene *F3*′*5*′*H* with its redox partner cytochrome P450 reductase gene *cpr* from *Catharanthus roseus* and successfully produced a hydroxylated flavonol quercetin from *p*-coumaric acid in *E. coli* by simultaneous coexpression of the fusion protein with 4CL, CHS, CHI, F3H, and FLS [110], which provides a guide to solve this kind of problem. To further improve the efficiency of the biosynthetic system, future research should be focused on screening key enzymes with high activities from various plants, mutation of genes encoding key enzymes to enhance their activities, and immobilization of the highly active enzymes to inert carriers.

Pectins and flavonoids are two distinctive classes of bioactive secondary metabolites presented in the fruit peels and used in food industry. The flavonoids can be divided into six major subclasses, including the flavanols, flavones, flavonols, flavanones, anthocyanidins, and isoflavones, and their flavonoid biosynthetic pathway has been largely elucidated. These natural small compounds possess a wide range of health-beneficial properties and can be derived by traditional plant extraction via organic solvents, chemical synthesis, and biosynthesis by constructing a microbial

cell factory or an *in vitro* multienzyme synthetic system.

The authors declare that they have no competing financial interests.

#### *Flavonoids and Pectins DOI: http://dx.doi.org/10.5772/intechopen.84960*

It is also much easier to purify desired products from this *in vitro* synthetic system than from the cell factory because of the simplicity of the components in the system. In addition, the strategy is highly cost-effective because of the cheap chemicals and recombinant proteins used in this system. More importantly, the system is easy to scale up and therefore possesses a huge industrialization potential. It also provides a guide for other secondary metabolites to produce economically. However, problems still exist in this production strategy. For example, due to the lack of P450-reductase function, prokaryotically expressed cytochrome P450 enzymes lose their enzymatic activities [109]. To achieve a functional expression, Leonard and colleagues fused a plant P450 enzyme gene *F3*′*5*′*H* with its redox partner cytochrome P450 reductase gene *cpr* from *Catharanthus roseus* and successfully produced a hydroxylated flavonol quercetin from *p*-coumaric acid in *E. coli* by simultaneous coexpression of the fusion protein with 4CL, CHS, CHI, F3H, and FLS [110], which provides a guide to solve this kind of problem. To further improve the efficiency of the biosynthetic system, future research should be focused on screening key enzymes with high activities from various plants, mutation of genes encoding key enzymes to enhance their activities, and immobilization of the highly active enzymes to inert carriers.
