Conventional and Emerging Extraction Processes of Flavonoids

: Flavonoids are a group of plant constituents called phenolic compounds and correspond to the nonenergy part of the human diet. Flavonoids are found in vegetables, seeds, fruits, and beverages such as wine and beer. Over 7000 ﬂavonoids have been identiﬁed and they have been considered substances with a beneﬁcial action on human health, particularly of multiple positive e ﬀ ects because of their antioxidant and free radical scavenging action. Although several studies indicate that some ﬂavonoids have provident actions, they occur only at high doses, conﬁrming in most investigations the existence of anti-inﬂammatory e ﬀ ects, antiviral or anti-allergic, and their protective role against cardiovascular disease, cancer, and various pathologies. Flavonoids are generally removed by chemical methods using solvents and traditional processes, which besides being expensive, involve long periods of time and a ﬀ ect the bioactivity of such compounds. Recently, e ﬀ orts to develop biotechnological strategies to reduce or eliminate the use of toxic solvents have been reported, reducing processing time and maintaining the bioactivity of the compounds. In this paper, we review, analyze, and discuss methodologies for biotechnological recovery / extraction of ﬂavonoids from agro-industrial residues, describing the advances and challenges in the topic.


Introduction
Flavonoids are natural pigments present in the plant or microbial sources and correspond to a specific group of chemical constituents called phenolic compounds [1,2].They are found in vegetables, seeds, fruit, and various fruits and alcoholic beverages [3].Flavonoids have important positive effects on human health especially due to their antioxidant and free radical scavenging.Although several studies have shown that some flavonoids have a pro-oxidant effect, they only occur at high doses, most of which confirm the existence of anti-inflammatory effects, antiviral or anti-allergic, and their protective role against cardiovascular disease, cancer, and various pathologies [2,4].
Flavonoids protect the human body from damage caused by oxidizing agents such as ultraviolet rays, environmental pollution, food chemicals, etc.The human organism cannot produce these chemicals in a protective manner, so they must be obtained by means of feed or as supplements.These compounds have been discovered by Nobel Prize winner Szent-György, who in 1930 isolated a substance, citrine, which regulates the permeability of the capillaries from the lemon peels.Flavonoids were first identified as vitamin P (because of the ability to increase capillary permeability) and vitamin C2 (because some flavonoids had similar properties to vitamin C) [1].However, the fact that flavonoids were vitamins could not be confirmed, and both names remain around 1950.Flavonoids contain in their chemical structure a variable number of phenolic hydroxyl groups and excellent properties of iron chelation and other transition metals, which give them a high antioxidant capacity; therefore, they play an essential role in the protection against oxidative damage and have therapeutic effects in a wide range of conditions, including heart disease ischemic, or atherosclerosis cancer [5][6][7].Antifree radical properties of flavonoids are primarily aimed at hydroxyl and superoxide radicals, highly reactive species involved in the onset of lipid chain peroxidation and described their ability to modify eicosanoid synthesis (with antiprostanoid and anti-inflammatory reactions) to prevent platelet aggregation (antithrombotic effects) and to protect low-density lipoproteins from oxidation [8,9].
In addition to its known antioxidant effects, flavonoids have other properties, including stimulation of communication through gap junctions, effects on the regulation of cell growth and induction of enzymes, detoxification such as dependent monooxygenase Cytochrome P-450, among others.[10].However, most of the biological properties of flavonoids are strongly determined by the mode of extraction for their recovery.Efforts have recently been reported to develop biotechnological strategies to reduce or eliminate the use of toxic solvents, reduce processing time, and maintain the bioactivity of the compounds.This paper examines, analyzes, and discusses the biotechnological methodologies and the recovery/extraction of flavonoids from agro-industrial residues, describing the advances and challenges in the field.

Flavonoids
Flavonoids are a type of polyphenolic compound, its chemical structure is varied but the general skeleton structure is composed of 15 carbones (C 6 -C 3 -C 6 ), which are grouped in two aromatic rings (A and B) connected by a 3-carbon bridge that gives rise to an oxygenated heterocycle (C) [11][12][13][14][15] (Figure 1).Flavonoids are derivatives of 1, 3-diphenylpropan-1-one and their biosynthetic pathway is the condensation of three malonyl-CoA molecules with one p-coumaroyl-CoA molecule to the intermediate chalcone [16,17].Flavonoids are water-soluble pigments present in the plant kingdom as secondary plant metabolites [2,18,19], which can be found specifically in the cytosol and stored in the plant cell vacuole [12,17].
These compounds have been discovered by Nobel Prize winner Szent-György, who in 1930 isolated a substance, citrine, which regulates the permeability of the capillaries from the lemon peels.Flavonoids were first identified as vitamin P (because of the ability to increase capillary permeability) and vitamin C2 (because some flavonoids had similar properties to vitamin C) [1].However, the fact that flavonoids were vitamins could not be confirmed, and both names remain around 1950.Flavonoids contain in their chemical structure a variable number of phenolic hydroxyl groups and excellent properties of iron chelation and other transition metals, which give them a high antioxidant capacity; therefore, they play an essential role in the protection against oxidative damage and have therapeutic effects in a wide range of conditions, including heart disease ischemic, or atherosclerosis cancer [5][6][7].Antifree radical properties of flavonoids are primarily aimed at hydroxyl and superoxide radicals, highly reactive species involved in the onset of lipid chain peroxidation and described their ability to modify eicosanoid synthesis (with antiprostanoid and anti-inflammatory reactions) to prevent platelet aggregation (antithrombotic effects) and to protect low-density lipoproteins from oxidation [8,9].
In addition to its known antioxidant effects, flavonoids have other properties, including stimulation of communication through gap junctions, effects on the regulation of cell growth and induction of enzymes, detoxification such as dependent monooxygenase Cytochrome P-450, among others.[10].However, most of the biological properties of flavonoids are strongly determined by the mode of extraction for their recovery.Efforts have recently been reported to develop biotechnological strategies to reduce or eliminate the use of toxic solvents, reduce processing time, and maintain the bioactivity of the compounds.This paper examines, analyzes, and discusses the biotechnological methodologies and the recovery/extraction of flavonoids from agro-industrial residues, describing the advances and challenges in the field.

Flavonoids
Flavonoids are a type of polyphenolic compound, its chemical structure is varied but the general skeleton structure is composed of 15 carbones (C6-C3-C6), which are grouped in two aromatic rings (A and B) connected by a 3-carbon bridge that gives rise to an oxygenated heterocycle (C) [11][12][13][14][15] (Figure 1).Flavonoids are derivatives of 1, 3-diphenylpropan-1-one and their biosynthetic pathway is the condensation of three malonyl-CoA molecules with one p-coumaroyl-CoA molecule to the intermediate chalcone [16,17].Flavonoids are water-soluble pigments present in the plant kingdom as secondary plant metabolites [2,18,19], which can be found specifically in the cytosol and stored in the plant cell vacuole [12,17].Flavonoids are classified according to differences in the structure of the heterocyclic C ring; these differences may be caused by the oxidation state and the degree of unsaturation of the heterocyclic ring (or the lack thereof in the case of chalcones).It has been estimated that the number of identified flavonoids exceeds 7000 and that the number of flavonoids continues to increase due to their important biological activities [17].Variations in the basic structure of flavonoids give rise to six different classes of this group of compounds: Isoflavones, flavanone, flavanone, flavan-3-ol, flavonol, and anthocyanidin, each of which has particular characteristics [18,20].

Flavanones
Flavanones (dihydroflavones) have a structure that differs in the lack of a double bond (C 2 -C 3 ) in the C-ring of the flavonoid structure.This type of flavonoid can be found in aromatic plants (such as mint), tomatoes, citrus (especially grapefruit) [12].Flavanones can be found in nature as forms of aglycones and glycosides, some examples of such compounds are naringenin, hesperetin, and eriodictyol [21].

Flavonols
Flavonols are called 3-hydroxyflavones and are the most commonly found flavonoids in the plant kingdom [22].A double bond between C 2 and C 3 and a hydroxyl group is included in their structures [13].Some of the most important phytochemical compounds that represent this group are as follows: Myricetin, quercetin, isorhamnetin, and kaempferol [12,22,23].They can be found in a variety of colors (from white to yellow).In nature, flavonols can be found in two forms: Glycosides and aglycone (quercetin and kaempferol) [12].

Flavones
Flavones can be found in all parts of the plants, above-and belowground, in vegetative and generative organs; stem, leaves, buds, bark, heartwood, thorns, roots, rhizomes, flowers, farina, fruit, seeds, and also in root and leaf exudates or resin.They result from the introduction of a double bond between C 2 and C 3 by the abstraction of two hydrogen atoms [16,18].Flavones are present in all major land-plant lineages.The plant species that contain flavones belong to over 70 different families in the plant kingdom [16].

Anthocyanins
Anthocyanins are primarily found in nature in glycosidic form.This type of flavonoid is responsible for plant pigment (such as blue, red, pink, and purple) by the formation of weak covalent bonding complexes with other organic compounds [23,24].More than 500 anthocyanins have been reported and are the product of methoxylation, hydroxylation, and glycosylation patterns in the B ring.The most representative compounds of this subclass of flavonoids are pelargonidin, cyanidin, and delphinidin [12].

Flavanols
Flavanols (flavan-3-ols) are also called catechins, which have a typical flavonoid structure but have different hydroxylation patterns of rings A and B and asymmetrical carbon stereochemistry of ring C (C 2 and C 3 ) [25,26].The catechins are classified into two groups; free catechins and esterified catechins [27], and constitute the most complex class of flavonoids due to their size, monomers (catechin), or polymeric forms (condensed tannins) [21].They can be found as the main ingredient in green tea [12].

Isoflavones
Another type of flavonoids, isoflavones are commonly referred to as phytoestrogens due to their considerable estrogen activity.They are characterized by the fusion of their ring B with the C 3 position of the ring C [21,[28][29][30].They are an important group in a variety of fields, such as medicine, cosmetics, and nutrition.These flavonoids can be found in plants of the Leguminosae family (soybeans, alfalfa sprouts, and red clover leaves) [30,31].

Isolation and Extraction Methods
There is a general methodology consisting of three stages for the isolation, extraction, and identification of phytochemicals from natural sources.Pretreatment or preparation of a sample is the first step in which the centrifuge, filtration, or drying process and others can be used.In the second stage, the extraction, isolation, and purification of flavonoid compounds from different plant samples are most notably.In this step, phytochemicals are extracted using processes such as soxhlet, maceration, water infusion, supercritical fluid extraction, solid microphase extraction, microwave extraction, ultrasound, autohydrolysis, etc.In the last step, the purified and extracted extracts are normally used for further study by chromatography techniques, usually involving the identification, quantification, and recovery of flavonoid compounds.
Details of each method, such as conventional and emerging methods used by a number of researchers for flavonoid extraction, are given in the following sections:

Conventional Methods
Flavonoid extraction and recovery have been booming over recent years because of population trends in healthier lifestyles and the integration of antioxidants into the diet.Therefore, several methods for extracting flavonoids to increase the extraction yields of these major bioactive compounds have been implemented.
Various extractive methods have been proposed, including maceration, percolation, hydro-distillation, boiling, reflux, soaking, and soxhlet [32].Soxhlet was the most commonly used method for the extraction of flavonoids due to its simplicity and ease of maintenance, low cost, and lower solvent content compared to other methods such as soaking, boiling, or maceration [14,33,34].Various solvents such as ethanol, methanol, benzene, chloroform, ethyl acetate, etc. have been tested in this extraction method to compare the effect on extraction yields [15,32].
In general, liquid-liquid or solid-liquid extraction is the most widely used process for the extraction of flavonoids.Although maceration and water infusion are conventional extraction processes, they are still used today [35,36].These methodologies have adopted the use of solvents such as ethanol, methanol, acetone and not just water for the extraction of bioactive compounds [14,37,38].These conventional extraction methods are characterized by the use of large amounts of solvent, lower extraction yields, and long extraction times compared to other methods.It has been reported that when extraction methodologies involve heat treatments, degradation in the chemical structures of the extracted flavonoids can result in a reduction in bioactivity [39].
Parameters such as time, particle size, type of solvents, mass to volume ratio, temperature, etc. have been evaluated in conventional extraction methods of flavonoid (Table 1) [40][41][42].The nature of the extracting agent (solvent) will affect the type of flavonoid extracted and will directly influence the biological activity of the recovered compounds.Of the solvents tested, ethanol and methanol are the most widely used for the extraction of flavonoids due to higher yields achieved in the recovery of flavonoids [43,44].The high demand for antioxidants gave way to the search for methodological alternatives that would increase the yield of flavonoid extraction and reduce process costs.In addition, it has been found that the methodologies implemented are cleaner and environmentally friendly.

Emerging and Advanced Methods
Two of the most widely used techniques for the extraction of flavonoids are emerging microwave (MAE) and ultrasound-assisted extraction (UAE) technologies.Table 2 shows some of the published works on the extraction of flavonoids using these emerging methods.

Microwave-Assisted Extraction
Microwave is an electromagnetic spectrum of radiation ranging from 300 MHz (radio radiation) to 300 GHz (infrared radiation).This heating technique uses microwave energy and is based on the direct effect of microwaves on dipole polarization and ion conduction molecules [105][106][107] (Figure 2).The extraction of flavonoids may be affected by a large number of parameters, among the most important of which are: Time, temperature, plant material-solvent ratio, solvent concentration, solvent polarity, irradiation, frequency of intensity, and microwave power [63,106,[108][109][110].
It has been reported that MAE allows for a significant reduction in the extraction times of a wide variety of compounds, also reduces the volumes of solvents used, and it has been shown that the extraction yields of bioactive compounds are superior to conventional methods such as maceration, Soxhlet, or heat reflux [66,106,112,113].Reduction of extraction times and the use of solvents are employed to improve the cost of extraction [39].
Microwave is an electromagnetic spectrum of radiation ranging from 300 MHz (radio radiation) to 300 GHz (infrared radiation).This heating technique uses microwave energy and is based on the direct effect of microwaves on dipole polarization and ion conduction molecules [105][106][107] (Figure -2).The extraction of flavonoids may be affected by a large number of parameters, among the most important of which are: Time, temperature, plant material-solvent ratio, solvent concentration, solvent polarity, irradiation, frequency of intensity, and microwave power [63,106,[108][109][110].It has been reported that MAE allows for a significant reduction in the extraction times of a wide variety of compounds, also reduces the volumes of solvents used, and it has been shown that the extraction yields of bioactive compounds are superior to conventional methods such as maceration, Soxhlet, or heat reflux [66,106,112,113].Reduction of extraction times and the use of solvents are employed to improve the cost of extraction [39].
The choice of solvent is not only important in this methodology; the dielectric properties of the solvent must be taken into account [106].The most commonly used solvents for MAE are ethanol and The choice of solvent is not only important in this methodology; the dielectric properties of the solvent must be taken into account [106].The most commonly used solvents for MAE are ethanol and methanol, both of which have shown the best extraction yields, although water has also shown positive effects [66,68,107].The efficacy of MAE will also depend on the type of flavonoid to be recovered.For example, the polarity of the flavonoid will be a very important parameter to consider; the type of solvent used will be the polarity of the desired recovery.Moreover, the solvent used in the extraction process may have an effect on the bioactivity of the recovered flavonoids [66].Apolar solvents such as dichloromethane, ethyl acetate, diethyl ether, chloroform are commonly used for the extraction of isoflavones, flavones, and methylated flavones due to their apolar nature.In contrast, solvents such as ethanol or methanol are used to extract polar flavonoids such as flavonoid glycosides and aglycones (Table 2A).

Ultrasound-Assisted Extraction
Ultrasound-assisted extraction is a technique that is used to rupture the plant material and extract the bioactive compounds with applications in industries such as food and pharmaceuticals [108].This technique is based on the phenomenon of acoustic cavitation, which consists of the formation of bubbles and the subsequent rupture, which causes the release of bioactive compounds, and this rupture depends on the extraction conditions [97,114] (Figure 3).
The cavitation effect produced by this methodology not only enables the destruction of the cell walls of the plant material but also promotes the reduction of the particle size that benefits the solvent-substrate interaction [97].There are many variables that can have an effect on flavonoid extractive processes and therefore on the number of experiments; in order to optimize a particular process, experimental matrices are usually used to perform the optimization process in order to determine the conditions that favor the recovery of the maximum flavonoid content.Most of the published works have opted to use the surface response methodology to achieve this objective [63].
Ultrasound-assisted extraction is a technique that is used to rupture the plant material and extract the bioactive compounds with applications in industries such as food and pharmaceuticals [108].This technique is based on the phenomenon of acoustic cavitation, which consists of the formation of bubbles and the subsequent rupture, which causes the release of bioactive compounds, and this rupture depends on the extraction conditions [97,114] (Figure 3).The cavitation effect produced by this methodology not only enables the destruction of the cell walls of the plant material but also promotes the reduction of the particle size that benefits the solvent-substrate interaction [97].There are many variables that can have an effect on flavonoid extractive processes and therefore on the number of experiments; in order to optimize a particular process, experimental matrices are usually used to perform the optimization process in order to determine the conditions that favor the recovery of the maximum flavonoid content.Most of the published works have opted to use the surface response methodology to achieve this objective [63].
The UAE increased the yield of bioactive compounds and the yield of flavonoids were shown to be variable depending on the method of extraction, as well as on the type of plant material (Table 2B).Mainly phenolic compounds and flavonoids, as well as the reduction of extraction times, and another of the advantages of this methodology is that it improves the biological properties of the extracts [115].The extraction of bioactive compounds depends on a variety of factors, such as the frequency used and usually between 20 kHz and 100 MHz [116], solvent selection, solvent concentration, solid-solvent ratio, temperature, and time extraction (Table 2B).
Among the flavonoid compounds that have been extracted and recovered using this methodology are the following: Rutin, narcissin, nicotiflorin, epicatechin, epicatechin gallate, The UAE increased the yield of bioactive compounds and the yield of flavonoids were shown to be variable depending on the method of extraction, as well as on the type of plant material (Table 2B).Mainly phenolic compounds and flavonoids, as well as the reduction of extraction times, and another of the advantages of this methodology is that it improves the biological properties of the extracts [115].The extraction of bioactive compounds depends on a variety of factors, such as the frequency used and usually between 20 kHz and 100 MHz [116], solvent selection, solvent concentration, solid-solvent ratio, temperature, and time extraction (Table 2B).
Among the flavonoid compounds that have been extracted and recovered using this methodology are the following: Rutin, narcissin, nicotiflorin, epicatechin, epicatechin gallate, catechin, procyanidin

Supercritical Fluid Extraction
Any substance at a temperature and pressure above its thermodynamic critical point is a supercritical fluid.Under these conditions, the properties of the fluids generate a high diffusivity and low viscosity of the solvents used to improve the process of transfer of the matter [120].Due to this, the SFE methodology (Figure 4) has reported flavonoid extraction yields much higher than those used in other techniques [121].
The most commonly used solvent in this extraction method is carbon dioxide (CO 2 ) due to its numerous advantages, such as that it is flammable, nontoxic, cheap, and very easy to remove due to its volatility [123,124].It is a strong solvent for supercritical extraction with all these features.
Certain advantages of this extraction methodology are low temperatures that maintain the integrity of the products, high volatility of the solvents which keep the waste low, the extraction is carried out without phase changes, easy separation of volatile and nonvolatile compounds.However, they present some limitations such as the difficult equilibrium between solute and solvent, may require other separation processes, high pressures hinder the continuous addition of solids to the extract, operating costs are high, equipment is low, maintenance cost is high, etc. [125,126].
Any substance at a temperature and pressure above its thermodynamic critical point is a supercritical fluid.Under these conditions, the properties of the fluids generate a high diffusivity and low viscosity of the solvents used to improve the process of transfer of the matter [120].Due to this, the SFE methodology (Figure 4) has reported flavonoid extraction yields much higher than those used in other techniques [121].The most commonly used solvent in this extraction method is carbon dioxide (CO2) due to its numerous advantages, such as that it is flammable, nontoxic, cheap, and very easy to remove due to its volatility [123,124].It is a strong solvent for supercritical extraction with all these features.
Certain advantages of this extraction methodology are low temperatures that maintain the integrity of the products, high volatility of the solvents which keep the waste low, the extraction is carried out without phase changes, easy separation of volatile and nonvolatile compounds.However, they present some limitations such as the difficult equilibrium between solute and solvent, may require other separation processes, high pressures hinder the continuous addition of solids to the extract, operating costs are high, equipment is low, maintenance cost is high, etc. [125,126].
Temperature is one of the most important factors in the process.In this methodology, the use of reduced temperatures is intended to keep the product as stable as possible.In one study, for example, the valorization of agro-industrial residues (expellers) from the extraction of soybean oil by pressing was analyzed.Extractions were carried out at 40 MPa and at 35 or 40 °C using CO2 as a solvent.Moreover, the expellers were impregnated with ethanol.The best results showed the flavonoids content of 65.0 and 31.3QE/100 gdm [127].On the other hand, flavonoids were extracted from Odontonema strictum leaves with supercritical carbon dioxide and ethanol.The effect of temperature (55-65 °C) on the total flavonoid content was optimized.The total flavonoid content and flavonoid recovery vary respectively from 99.33 to 247.78 mg/g of dried extract and 10.68-18.92mg/g of dried Temperature is one of the most important factors in the process.In this methodology, the use of reduced temperatures is intended to keep the product as stable as possible.In one study, for example, the valorization of agro-industrial residues (expellers) from the extraction of soybean oil by pressing was analyzed.Extractions were carried out at 40 MPa and at 35 or 40 • C using CO 2 as a solvent.Moreover, the expellers were impregnated with ethanol.The best results showed the flavonoids content of 65.0 and 31.3QE/100 gdm [127].On the other hand, flavonoids were extracted from Odontonema strictum leaves with supercritical carbon dioxide and ethanol.The effect of temperature (55-65 • C) on the total flavonoid content was optimized.The total flavonoid content and flavonoid recovery vary respectively from 99.33 to 247.78 mg/g of dried extract and 10.68-18.92mg/g of dried leaves powder [128].In another study, supercritical CO 2 extraction with/without ethanol from Citrus unshiu peels was examined at a temperature of 59.85 • C and a pressure of 30 MPa.The best results obtained were that the extracts, including nobiletin, increased the concentration of ethanol in supercritical CO 2 and increased the time of extraction.On the other hand, the role of pressure in these methods is very important and depends on the solvent used.For example, an effective method of extracting Medicago sativa using enzyme-assisted supercritical fluid was developed in another study.The design of the Box-Behnken was chosen to optimize the extraction process parameters, including pressure (100-300 bar).Optimal extraction parameters for total polyphenol content were: 68 • C, 205 bar, and 15.5% for temperature, pressure and co-solvent content, respectively.This methodology provides effective enzyme-assisted supercritical fluid extraction for the enhanced release of polyphenol compounds [129].Supercritical fluids are a more efficient, safer, and environmentally friendly method for extracting and recovering flavonoids for the purpose of bioactive compounds study (Table 2C).Enzyme-assisted extraction is a promising alternative to conventional extraction methods where a high amount of solvent is used.The advantages of this method are regioselectivity and specificity of enzyme action, their ability to catalyze reactions in aqueous solutions under mild conditions [131].Furthermore, the application of enzyme is an environmentally friendly method for the extraction of flavonoids due to a decrease in solvent quantity in order to reduce extraction times and increase extraction yield and quality of flavonoids.Enzymes and microorganisms may also be used for pretreatment in different agro-industrial and food industries.

Enzyme and Microorganism-Assisted Extraction
Enzymes obtained from fungi, bacteria, vegetable extracts and animal organs, such as pectinases, different glucanases, hemicellulases, cellulases, etc., in mixtures or alone, break down the cell wall by hydrolysis of biopolymeric components to increase the permeability of the cell wall and also increase the yield of flavonoids and other physiologically active extractants [132,133].
Hydrolytic enzymes can break down the cell wall of polysaccharides to improve intracellular release.
Several authors have utilized enzymatic treatments with commercial preparations for the extraction of flavonoids from plant material (Table 3).These commercial enzymes usually have one or more main hydrolytic activities and a number of side activities.Commercial pectinases have been used for enzyme-assisted extraction of black currant phenols (Ribes nigrum) juice press residues [135].Mixtures of pectinases and cellulases have been used for the extraction of polyphenols from grape pomace [136,137].A combination of enzymatic hydrolysis and ultrasonic-assisted extraction was used for the extraction of flavonoids from pigeon pea (Cajanus cajan) leaves [138], celery (Apium graveolens) leaves [139], shepherd's purse (Capsella bursa-pastoris) pulp [140], and mulberry (Morus nigra) must [141].A set of combinations of different extraction methods (viz.enzymatic hydrolysis, ultrasonic, and microwave-assisted extraction) that were used for the recovery of flavonoids from Nitraria tangutorun juice by-product [142].
Special attention must be paid to the presence of undesirable enzyme activities during the enzyme-assisted extraction of flavonoids.These activities may be caused by endogenous enzymes of plant material or by side activities of commercial preparations.Polyphenol oxidases may induce coupled oxidative browning reactions; β-glucosidases, β-galactosidases, and α-l-arabinosidases may hydrolyze native glycosylated anthocyanins and cause unstable aglycons [135].For example, Kammerer et al. (2005) [136] observed a low yield of anthocyanin recovery (2.9%) during enzyme-assisted extraction of polyphenols from grape pomace (Vitis vinifera) at the pilot plant level.This low yield was associated with the action of endogenous enzymes.In subsequent research, thermal inactivation of endogenous enzymes prior to aqueous extraction and enzyme treatment allowed 63.6% of anthocyanins to be recovered from grape pomace [137].Landbo and Meyer (2001) [135] found that two commercial pectinases had a negative effect on the recovery of anthocyanins during enzyme-assisted extraction of phenolic compounds from black currant (Ribes nigrum) pomace.This negative effect was associated with the presence of glycosidase activities in enzyme preparations.On the other hand, Xu et al. (2013b) [145] used the activity of glycosylase present in commercial enzyme preparations to improve the extraction of bioactive compounds from two medicinal plants (Glycyrrhizae radix and Scutellariae radix).Bifunctional enzymes were used for the simultaneous degradation of the cell wall and deglycosylation of native flavonoid glycosides.
The parameters to be taken into account for increasing the extraction yield of flavonoids are treatment time, pH, and temperature, as well as the enzyme quantity in relation to the concentration of the substrate.Various studies reported the optimization of these parameters [143,144].
There are also disadvantages of enzymatic methods, which have been reported in many reports: To date, the enzyme tested has not been able to achieve complete plant cell wall hydrolysis due to the major difficulties of scale-up enzyme-assisted extraction at industrial level and the relatively high cost of biocatalyst for large volumes of raw materials [132].The use of microorganisms as enzyme producers may replace the use of food-grade enzymes in the extraction of flavonoids.However, their growth and activity are sensitive to changes in environmental conditions (temperature, percentage of dissolved oxygen, agitation rate, design of reactors, and availability of nutrients) [147].

Microbial Biotransformation
Biotransformation of flavonoids to increase the biological activity of the recovered compounds is a trend.Biotransformation is a process in which the chemical structure of the compounds is modified by the use of microorganisms.The objective of biotransformation is to produce fine chemical compounds (high added value) that are difficult to produce by chemical synthesis under low-severity reaction conditions.These processes have good production yields and the recovery of flavonoids.The main biotransformations reported in flavonoids are as follows: Dehydroxylation, dehydrogenation, hydrogenation, glycosylation, O-methylation, O-demethylation, deglycosylation, cyclization, sulfation, and carbonyl reduction [148].This section discusses some biotransformation studies on flavonoids using filamentous fungi and bacteria.
Filamentous fungi and 20 strains of Streptomyces for the production of flavonoids were evaluated and two derivatives of quercetin were obtained from Beauveria bassiana ATCC7159 in the bioconversion of quercetin.Furthermore, the bioconversion of rutin was obtained by Cunninghamella echinulata ATCC 9244 rutin sulfate, rutin glucuronide, and rutin methylation.This biotechnology method was appropriate to produce biologically-active flavonoids [149].In another study, the biodegradation of isoflavones into 4 -fluoroisoflavone were evaluated by Aspergillus niger and Cunninghamella elegans strains and obtained more than 20 metabolites.A. niger was the microbial strain that has the most ability to degrade isoflavones which could be used as bioactive compounds [150].Furthermore, the biotransformation of icariin, epimedin C, epimedoside A, epimedin A, and epimedin B were evaluated from the Epimedium koreanum plant using Cunninghamella blakesleana.This process generated flavonoids that posed potential applications in the pharmaceutical and food industries [151].
A new method was developed to produce genistein from roots of pigeon pea (Cajanus cajan) using immobilized strains of Aspergillus oryzae and Monacus anka.This biotransformation method was a good alternative to the production of genistein from plants with food industry potential [152].Different filamentous fungi used in solid-state fermentation were evaluated for the biotransformation of phenolic compounds in cauliflower leaves.A. sojae strain was best suited to high yields of flavonoids, including kaempferol-derived metabolites.This bioprocess was proposed as an alternative for the development of the concept of bio-refinery and the use of agricultural by-products [153].In another study, a process of biotransformation of phenolic compounds from citrus waste using solid-state fermentation by Peacilomyces variotii was developed, where remarkable production of naringenin and hesperetin was achieved and the antioxidant capacity increased to 73%.These compounds are of high added value that can be used in the food sector [154].
Moreover, a strategy for the biodegradation pathway from tyrosine to the production of fisetin using Escherichia coli has been developed.The production of this flavonoid is of great interest because it has different biological properties for human health, such as antiviral and anticancer [155].In another study, the biotransformation of soy isoflavones in ortho-hydroxyisoflavones was evaluated using CYP105D7 from Streptomyces avermitilis MA4680 and expressed in Pichia pastoris.This study provides evidence of the great potential of the use of genetic microorganisms for the production of isoflavonoids for food industry applications [156].Puerarin catalyzed by Bacillus cereus NT02 was evaluated for its biotransformation.The results showed that puerarin phosphorylation innovations in medicinal chemistry have particular importance [157].

Enzyme-Catalyzed Transformation
Flavonoids are characterized by their low solubility and stability in aqueous and lipid phases.For instance, aglycones are less soluble than their derivatives.Aglycones are readily absorbed by passive diffusion through biological membranes, while flavonoid glucosides can be introduced into cells by means of a sodium-dependent glucose transporter 1.Therefore, deglycosylation is important for the assimilation of dietary flavonoids [158].They may be modified chemically, enzymatically, or chemo-enzymatically to enhance these properties.
Glycosylation and acylation are the most important transformations of flavonoids catalyzed by enzymes.Glycosylation allowed flavonoids to enhance their hydrophilic character by adding sugars, while acylation makes them more hydrophobic due to the combination of fatty acids.Chemical acylation is not regioselective [159] and results in the modification of phenolic groups responsible for the antioxidant activity of flavonoids [158].The enzyme groups used for flavonoid transformation are presented in Table 4. Cyclization of chalcone to form flavanone, transformation of chalcone and 6 -deoxychalcone into (2S)-naringerin and (2S)-5-deoxyflavanone.
Soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. [161]

Laccase
Laccase from Myceliophthora Synthesis of a flavonoid polymer and high molecular fraction of extracted flavonoids from rutin as substrate in the mixture of methanol and buffer.Oxidation of catechin in the presence of gelatin and synthesize the gelatin-catechin conjugate.
[ 162,163] Lipase Candida antarctica lipase B Acetylation only on the primary 6 -OH of the isoquercitrin glucose and the secondary 4 -OH of the rutin rhamnose were expected to be acetylated. [164]
[ 146,165,166] Pectinolytic and Cellulolytic Enzymes Hydrolysis of main-and side-chain of polysaccharides, and glycosidase activities.

Protease
Alkaline protease from Bacillus subtilis Synthesis of 3"-O-substituted vinyl rutin esters in pyridine.

Proteases
Hydrolytic and synthetic functions.Enzymatic transformations of flavonoids were affected by the type, origin and concentration of enzymes, nature of flavonoids, donor and optimal conditions (temperature, substrates, and solvent).

Subtilisin (serine protease)
Flavonoid ester synthesis, the selective rutin acylation in organic solvents with excellent selectivity.The structure of the sugar moiety affected the regioselectivity.Lipase (Table 4) catalyzed flavonoids acylation with phenolic acids leads to increased solubility, stability, and antioxidant activity of flavonoids in different media [176].In addition, the presence of electron-donating or withdrawing substituents in the aromatic ring of flavonoids appears to be essential for their activity in the central nervous system as anxiolytic, anticonvulsant, and sedative and skeletal muscle relaxant drugs.
Chemical modifications are complex and laborious work that requires specific conditions.Therefore, enzyme modification can be a very promising alternative technique in this way.It is a well-known enzymatic halogenation of organic compounds by means of chloroperoxidase (EC 1.11.1.10).Chloroperoxidase from Caldariomyces fumago (Table 4) and whole microbial cells were applied to halogenate the flavones, naringenin, and hesperetin, at C-6 and C-8 in the presence of chloride and bromide ions [168].Biomodified compounds have shown similar properties compared to derivatives obtained by chemical modification using highly aggressive agents such as molecular halogens and hypohalous acid.
The use of different enzymes (Table 4) has been studied in order to find the most potent biocatalyst for the selective transformation of flavonoids (acylation, deacylation, etc.).Applications of thermostable enzymes have been reported [166].Enzyme immobilization has been performed to increase enzyme stability, facilitate enzyme reuse, and product insulation [177].
The regioselective synthesis of phloridzin-6'-O-cinnamate has been carried out using Candida antarctic lipase B immobilized with a macroporous acrylic resin [178,179].Different enzymes have been used in immobilized forms to reduce the cost of enzymatic modifications of structural flavonoids due to their advantages such as easy isolation and re-use, increased enzyme stability, and regioselectivity in nonaqueous media [164].

Human Health and Biological Properties
There is a strong link between a high intake of flavonoids and low cardiovascular disease, neurodegeneration, and cancer in the population in which a food that includes high levels of these phytochemicals is included in a daily diet [18,22,182].These properties are based on protein interaction (enzyme, factor transcription, and receptor transcription) [22].The use of herbs with a high content of flavonoids has been used as part of traditional remedies to treat various diseases that improve the immune system, as shown by antioxidants, anti-inflammatory, anti-allergenic, and antithrombotic drugs (Figure 5) [19,183,184].There are some reports indicating the pro-oxidant activity of flavonoids [185].Flavonoids generally act to protect plants from UV-B radiation and have the potential to reduce oxidative damage, act as photoreceptors, and have the power to attract flower pigment pollinators and protect against pathogen attack [19,23,186].In particular, isoflavones have shown a variety of effects on human health, especially in women's health, such as the prevention and reduction of climate symptoms (such as hot flushes).The control of diseases such as diabetes, osteoporosis, and breast cancer has also been reported to be associated with the consumption of food rich in isoflavones [28,30,31].In addition, even for prostate cancer isoflavones have shown their chemical-prevention effect [29].

Concluding Remarks
Flavonoids are a category of compounds with diverse biological activities that are of interest to various industrial areas, such as food, health pharmaceuticals, and cosmetics.Due to consumers demands today for products that benefit their quality of life substantially.Several extraction methods for the recovery of these flavonoids have been proposed for its wide range of applications.Today, microwave, ultrasound, and supercritical fluid technologies are among the most used methods of flavonoid extraction by most researchers worldwide.In addition, the use of biotechnological alternatives has attracted the attention of research to finding cleaner and more efficient ways to recover flavonoids.These methodologies have been distinguished from conventional methods by a marked increase in extraction yields and the displacement of organic solvents by "green" solvents, as well as a significant decrease in extraction times.Most studies have reported the extraction of flavonoids from vegetable sources such as fruits, seeds, roots or by-products of food, and/or beverage processing such as peels.Although progress has been made in this area, it is necessary to redefine the extractive techniques, since the majority is focused on increasing the yield of total flavonoids, leaving aside the purity of the extracts.The acquisition of extracts and/or purer flavonoid compounds would allow us to know more precisely the biological activities and thus to understand the mechanisms of action and the effects on their consumption, allowing for greater diversification of their applications and generating more knowledge in this area.In particular, isoflavones have shown a variety of effects on human health, especially in women's health, such as the prevention and reduction of climate symptoms (such as hot flushes).The control of diseases such as diabetes, osteoporosis, and breast cancer has also been reported to be associated with the consumption of food rich in isoflavones [28,30,31].In addition, even for prostate cancer isoflavones have shown their chemical-prevention effect [29].

Concluding Remarks
Flavonoids are a category of compounds with diverse biological activities that are of interest to various industrial areas, such as food, health pharmaceuticals, and cosmetics.Due to consumers demands today for products that benefit their quality of life substantially.Several extraction methods for the recovery of these flavonoids have been proposed for its wide range of applications.Today, microwave, ultrasound, and supercritical fluid technologies are among the most used methods of flavonoid extraction by most researchers worldwide.In addition, the use of biotechnological alternatives has attracted the attention of research to finding cleaner and more efficient ways to recover flavonoids.These methodologies have been distinguished from conventional methods by a marked increase in extraction yields and the displacement of organic solvents by "green" solvents, as well as a significant decrease in extraction times.Most studies have reported the extraction of flavonoids from vegetable sources such as fruits, seeds, roots or by-products of food, and/or beverage processing such as peels.Although progress has been made in this area, it is necessary to redefine the extractive techniques, since the majority is focused on increasing the yield of total flavonoids, leaving aside the purity of the extracts.The acquisition of extracts and/or purer flavonoid compounds would allow us to know more precisely the biological activities and thus to understand the mechanisms of action and the effects on their consumption, allowing for greater diversification of their applications and generating more knowledge in this area.

Figure 1 .
Figure 1.Basic chemical structures of flavonoids and their different class.
There are several different techniques for the extraction of flavonoids, including conventional solid-liquid extraction, pressurized fluid extraction, pressurized hot water extraction.Supercritical fluid extraction, MAE, UAE, and pulsed electrical field extraction are among the most sustainable alternatives to these traditional methods [130].The process of extraction of flavonoids is usually performed using organic solvents mixed with or without water.The extraction can be controlled by different parameters, such as the selection of extraction procedures, as well as the flow rate used for extraction, solvent and temperature, pressure and time.In addition, other techniques have yielded similar results; these techniques involve the use of microorganism-and enzyme-assisted extractions.

Processes 2020, 8 , 28 Figure 5 .
Figure 5. Different biological properties of plant flavonoids for human health and disease.

Figure 5 .
Figure 5. Different biological properties of plant flavonoids for human health and disease.

Table 1 .
Summary of studies of isolation and extraction of flavonoids from different plant sources.

Table 2 .
Studies on emerging methods of isolation and extraction of flavonoids from different plant sources.

Table 3 .
Studies on enzyme assisted isolation and extraction of flavonoids from different plant sources.

Table 4 .
Flavonoids transformation catalyzed by different enzymes.