Food Bioprocess Technol (2012) 5:409–424

DOI 10.1007/s11947-011-0573-z

REVIEW PAPER

Microwave-Assisted Extraction of Flavonoids: A Review

Winny Routray & Valérie Orsat

Received: 5 January 2011 / Accepted: 29 March 2011 / Published online: 12 April 2011
# Springer Science+Business Media, LLC 2011

Abstract Flavonoids are major bioactive compounds for treatment or prevention of certain diseases (Bravo and
known to be beneficial against many chronic diseases such Mateos 2008). Some of the most important bioactive
as cancer, cardiovascular disorders, and inflammation. Food compounds from plant sources are polyphenolic com-
products obtained from plants are key sources of flavonoids pounds and mainly flavonoids. Flavonoids are generally
for humans. However, during different food-processing antioxidants and act as free radical scavengers as they are
steps, flavonoids are lost in remarkable amounts. Supple- potential reducing agents and protect from oxidative
mentation of flavonoids as a food additive will help in reactions taking place inside the body. They act against
retaining the required health-beneficial amount of flavo- many chronic diseases such as cancer (Birt et al. 2001;
noids in the diet, and extraction is an important step in the Middleton et al. 2000), inflammation (Manthey et al. 2001;
preparation of food additives. Microwave extraction is one Middleton et al. 2000), cardiovascular (Kris-Etherton and
of the most advanced extraction methods, which has the Keen 2002; Mennen et al. 2004), and neurodegenerative
potential of playing a major role in flavonoid extraction disorders (Lu et al. 2010; Moosmann and Behl 2002). They
and analytical quantification. The present paper reviews are beneficial against many other health problems such as
the potential application of microwave extraction for osteoarthritis (Farid et al. 2010) and ocular disorders
flavonoids and the advantage of microwave-assisted (Majumdar and Srirangam 2010) and act as phytoestrogen
process over the other extraction processes generally (Cherdshewasart et al. 2007; Han et al. 2002) and
employed for extraction. antithyroidal compound (Chandra and De 2010; Chandra
et al. 2010). Flavonoids are present in all the higher plants
Keywords Flavonoids . Microwave-assisted extraction . and in some animals where it is suspected to be derived by
Extraction the consumption of foods of plant origin (Bravo and
Mateos 2008). Flavonoids are beneficial to humans and
also to the plants. They play a significant role in the plant
Introduction growth, reproduction, and plant’s defence mechanism
against factors such as pathogens and UV radiations
Bioactive compounds are the extra-nutritional constituents (Treutter 2006). They occur in many fruits and vegetables
present in some foods which quantitatively constitute only a which are a large part of a balanced human daily diet.
small part of them (Kris-Etherton et al. 2002). Several Depending on the constituents of the diet, flavonoids intake
bioactive compounds are used as nutraceuticals, which can can vary in the range of 50–800 mg per day (Stalikas
be defined as compounds having health-beneficial effects 2007).
In the last few decades, increasing health consciousness
has attracted the attention of food scientists towards
W. Routray : V. Orsat (*) flavonoids. Fortification with flavonoids in processed food
Bioresource Engineering Department, Macdonald Campus,
is encouraged to ensure adequate daily intake to enhance
McGill University,
Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9 the immune system. The food-processing industry has
e-mail: [email protected] known a great expansion due to the increasing population
410 Food Bioprocess Technol (2012) 5:409–424

demand for healthy and convenient processed foods. This structural variations, which lead to other variations (varia-
has led to an increase in the amount of industrial by- tions in physical and chemical properties) between the
products and wastes such as peels, pulp, and seeds. These different classes and within the classes, depend on the
food industry wastes can be used for the extraction of the degree and pattern of hydroxylation, methoxylation, pre-
flavonoids they contain as a supplement to the direct nylation, or glycosylation (Stalikas 2007). The structures of
sources which are fruits and vegetables themselves. There different flavonoids with their substitution patterns have
might be a scope for synthesis of these compounds but use been discussed in detail in different reviews and book
of a natural source for extraction and fortification is the chapters (Bravo and Mateos 2008; Stalikas 2007) along
preferable option from a market acceptability point of view. with different sources of foods rich in flavonoids. The
Therefore, extraction represents an important contributing general source of food flavonoids are plant sources with
element to the development of processed foods and some exceptions. For instance, flavonols and flavones are
nutraceutical food ingredients. Because of the inherent also present in algae and fungi. Studies have shown that the
diversity of flavonoids, it is difficult for the analytical aerial parts (Bravo and Mateos 2008), especially outer
chemists and biochemists to rapidly and systematically layers (leaves, fruit peels, stems, and seed coats) of the
quantify flavonoids (Stalikas 2007). The common methods plants are rich in flavonoids, which has been supported by
of extraction of flavonoids include conventional methods the fact of relation of flavonoid synthesis with sunlight
such as fluid extraction, soxhlet extraction, and ultrasound (Herrmann 1976). The different pathways of biosynthesis
extraction or modern methods such as supercritical and of flavonoids have been described in detail in the book
subcritical extraction and pressurized liquid extraction. “The Flavonoids” (Harborne et al. 1975). They are mainly
Scientists are exploring other possible methods which are derived by a combination of the products of the shikimate
more environment friendly, using less energy and less pathway and acetyl CoA carboxylase reaction. Carbohy-
solvent while producing higher yields. In the present drates in the Calvin cycle form shikimic acid, from which
scenario, to satisfy these requirements, microwave-assisted phenylalanine is formed. These get converted to coumaryl
extraction can be a potential option. This paper, thus, CoA through the phenylpropanoid pathway. Acetyl CoA
explores the possibility of using microwave extraction for carboxylase reaction converts acetyl CoA to malonyl CoA.
extracting flavonoids. It presents a brief discussion regard- Coumaryl CoA and malonyl CoA combine to form
ing flavonoids, their general methods of extraction, chalcones from which different flavonoids are derived.
microwave-assisted extraction and quantification, and The pathways of formation of different flavonoids have
finally discusses the possible benefits obtained by micro- been discussed by several authors (Stafford 1990; Hendry
wave extraction for the production of flavonoids plant and Houghton 1996). Aromatic amino acids such as phenyl
extracts and its present situation in this context. alanine, tyrosine, and malonate have also been reported as
the compounds from which flavonoids are formed (Stalikas
2007).
Flavonoids As mentioned before, flavonoids have many beneficial
health effects and are an important part of the food-
Flavonoids are secondary plant metabolites, present almost nutraceutical industry. Fruits, vegetables, and different
in all growing parts of the plants. They have been reported beverages are the primary sources of flavonoids. However,
to be the most abundant plant pigment along with during storage and many processing techniques, the
chlorophyll and carotenoids (Stalikas 2007). The flavan flavonoid content diminishes due to different factors of
nucleus, 1,3-diphenylpropane (Fig. 1) is the basic skeletal degradation. In case of storage for instance, several
structure of flavonoids which consists of two aromatic rings enzymatic reactions are taking place in different food
linked by a three-carbon structure. This structure forms an systems which affect the flavonoids content. It has been
oxygenated heterocycle except in the cases of chalcones reported that most of the flavonoid content in fruits and
and dihydrochalcones. Flavonoids can be grouped into vegetables is present in the surface layer, i.e., the skin, so it
different classes. Major classes of flavonoids based on
abundance in food are flavonols and flavones, isoflavones,
flavanones, and flavandiols, anthocyanins and proantho- Fig. 1 Flavan nucleus, the basic
cyanidins, and catechins and their structures are presented skeletal structure of flavonoids
in Table 1 (Bravo and Mateos 2008). Other classes of
flavonoids include flavan-3-ols, anthocyanidins, chalcones,
and other biosynthetic intermediates of the flavonoid
biosynthesis such as aurones, biflavonoids, and dihydro-
chalcones (Bravo and Mateos 2008; Stalikas 2007). The
Food Bioprocess Technol (2012) 5:409–424 411

Table 1 Skeletal chemical structures of major classes of flavonoids

Classes of Basic chemical structure
flavonoids
Flavonols

Flavones

Catechins

Isoflavones

Flavanones

Anthocyanins

Proanthocyanidins

Anthocyanidins

Chalcones

Data adapted from Bravo and Mateos (2008). Substituents are –OH, –OCH3, glucose, gallate, rhamnose, rhamnoglucoside, rutinoside,
galactoside, or neohesperidoside. For chalcones the substituents could be some of the above mentioned groups and some halides
412 Food Bioprocess Technol (2012) 5:409–424

is very easy to lose those flavonoids as simple peeling can mechanical stirring, and continuous rotary extraction
significantly reduce the content. Other processing steps, (Guillen et al. 1996) are some other methods which can
such as heat treatment, maceration and juice extraction be applied to increase furthermore the molecular interac-
decrease the flavonoid content further due to enzymatic and tions (Stalikas 2007). From various extraction studies, it is
thermal breakdown. Synthesis or extraction of flavonoids observed that the choice of pretreatment is not coherent,
from natural sources and their use for the fortification of which can be explained from the complexity of flavonoids’
processed foods would help to maintain high flavonoid properties. Polarity, acidity, presence of hydroxyl groups,
contents in the foods we consume. This would lead to more and aromatic rings are some of these properties (Stalikas
options for both producers and consumers along with 2007). Furthermore, the biological matrix is very complex
maintaining the expected nutritional food quality. and it differs with each source; the biological matrix of one
variety of grapes is different from another which also
differs with the conditions of plant growth.
Conventional Methods of Extraction The most appropriate method of extraction depends on
the source and the type of flavonoid to be extracted. The
The whole extraction procedure can be divided into selection criterion of the best extraction method is based on
different steps as illustrated in Fig. 2—pretreatment, the highest recovery, for which the degradation of the
extraction, isolation, and purification (Stalikas 2007). The compounds due to the different process steps should be
production of functional/nutraceutical food ingredients may minimum. Some forms of flavonoids (aglycones) are
also involve the additional step of encapsulation, which reported to be more easily absorbed from the diet as a
may involve simple drying or addition and mixing of bioactive compound than the other forms of flavonoid
different stabilizers followed by drying. It is evident that for glycosides (Stalikas 2007). So along with the parameters
analytical chemistry in the determination of flavonoid involved, decision regarding the choice of extraction
content as well as for production of flavonoids as food method also depends on the end product required. There
ingredients, extraction is an important process step. The have been numerous reviews and research on the advance-
pretreament steps can include maceration, homogenisation, ment of different extraction techniques with comparative
grinding, milling, or drying (generally freeze drying to discussions between them (Camel 2001; Dai et al. 2010;
prevent degradation of flavonoids). Drying can be useful Kaufmann and Christen 2002; Raynie 2006; Stalikas 2007).
for increasing yield per unit weight of raw material, Solvent extraction is the most common method of
increasing storage life of the raw material, decreasing space extraction of flavonoids. Different types of solvent extrac-
requirement and in some cases for ease in further process- tion methods are used currently, out of which hot water
ing. Maceration, grinding, milling, and homogenisation bath extraction (de Rijke et al. 2006; Søltoft et al. 2009)
increase the contact surface area between the solvent and and soxhlet extraction method (Kalia et al. 2008) are the
the sample containing the solute; the solute being the most common for extraction of bioactive compounds
flavonoid contained within the cellular structures of the including flavonoids. For comparison of advanced methods
biological samples. These pretreatment steps lead to the in terms of their yield, the soxhlet method is still used as a
breakdown of cellular structures which enhances further the standard in almost all cases (Fan et al. 2010; Macdonald
yield of the bioactive compounds. Use of vortexing et al. 2010; Pan et al. 2010; Zhu et al. 2010). Methanol or
followed by centrifugation (Montedoro et al. 1992), acetone is used as the solvent for soxhlet extraction of

Pretreatment: Extraction: Isolation and Purification:

• Maceration • Hot water bath • Solid phase extraction

• Grinding • Soxhlet extraction • Acidity based
• Milling • Sonication assisted fractionation
• Homogenisation extraction • Column chromatography
• Mechanical stirring • Pressurized fluid extraction
• Drying • Accelerated fluid extraction
• Subcritical fluid extraction
• Supercritical extraction
• Microwave assisted
extraction

Fig. 2 Different steps that may be involved in the extraction of flavonoids

Food Bioprocess Technol (2012) 5:409–424 413

flavonoids. The process of soxhlet extraction used in the thus decreasing the extraction time remarkably, even up to
nutraceutical industry has been briefly described by Wang less than 50% of the time required in most other methods
and Weller (2006). The usefulness and the prospects of (Qian et al. 2009). Due to the application of high pressure
soxhlet extraction method as compared with modern and high temperature, the extraction rate is increased with
methods of extraction have been discussed in detail by the increased diffusivity of the solvent into the biological
Luque de Castro and Garcia-Ayuso (1998). The main matrix. This process needs less solvent and the oxidative
drawback in the use of soxlet extraction is the combination degradation of the flavonoids is lower, as the process is free
of long hours of extraction and high temperature which of oxygen and light (Raynie 2006; Stalikas 2007; Wang and
affect the recovery of mainly thermolabile components, Weller 2006).
which is of concern in the case of flavonoids (Wang and Accelerated solvent extraction is a type of pressurized
Weller 2006). The use of large amounts of solvents is also a solvent extraction where the temperature range is
problem in terms of sustainability and protection of the between 50 to 200 °C, pressure generally varies from
environment. 10 to 15 MPa, and the solvent remains below critical
Other solvent extraction method such as sonication- condition, in its liquid state (Wang and Weller 2006).
assisted extraction is also used for extraction of flavonoids The solvent, in the case of accelerated solvent extraction,
(Maksinovic et al. 2005). Use of ultrasound (sound waves is usually an organic solvent (Abourashed et al. 2002;
which have frequencies higher than 20 kHz) can disrupt Waksmundzka-Hajnos et al. 2008). Water can also be used
plant cell walls with subsequent increase in solvent as the extraction medium; where application of high
penetration, which helps in obtaining a higher extraction pressure allows the solvent to be used above its boiling
yield. Ultrasound-assisted extraction can be a technique of point. As mentioned by Raynie (2006), water loses its
choice for thermolabile components as the operating effective polarity at these high temperatures which allows
temperature can remain low during this process, thus it to act as a solvent for both polar and non-polar
maintaining extract quality (Wang and Weller 2006). compounds. This method is also known as subcritical
Ultrasounds have been applied in the case of extraction of extraction or pressurized hot water extraction. Hot water
flavonoids from different sources such as medicinal plants extraction has been efficiently used for the extraction of
like Selaginella doederleinii (Li et al. 2010) and Citrus flavonoids (Ibanez et al. 2003; Rodriguez-Meizoso et al.
aurantium (Yang et al. 2010), food-processing waste such 2006).
as litchi pericarp (Rao 2010), and longan fruit pericarp Supercritical fluid extraction is another popular method
(Prasad et al. 2010). applied for extraction of many bioactive compounds. The
supercritical state is reached when both the temperature and
pressure are raised above their critical value. This increases
Advanced Methods of Extraction the fluidity which accounts for the increased mass transfer.
It is a clean method, considered environmental friendly as
Many existing methods of extraction have some major CO2 is generally used as the solvent. It is a very efficient
drawbacks such as a long process time, which eventually method for extraction of non-polar compounds, as CO2 is a
leads to thermal degradation of the flavonoids (de Rijke et non-polar solvent, however, to increase the solubility of
al. 2006), and high solvent requirements, raising process polar compounds, solvent such as methanol, ethanol,
costs and reducing environmental sustainability. The pres- acetonitrile, acetone, water, ethyl ether, and dichlorome-
ence of oxygen and light during the extraction contribute in thane can be added (Wang and Weller 2006). This increases
additional degradation of the target compounds. Some of the extraction selectivity of this extraction method. As
the newer extraction methods such as pressurized fluid evidenced from the literature, the modification of the
extraction, accelerated fluid extraction, subcritical fluid solvent composition has lead to the successful extraction
extraction, and supercritical extraction are helpful in of flavonoids from biological matrices (Catchpole et al.
mediating associated extraction problems along with 2004; Liza et al. 2010; Yu et al. 2007), however, for some
increased extracted yields. Wang and Weller (2006), very polar compounds, the extraction efficiency of super-
Stalikas (2007) and Raynie (2006) have discussed in details critical extraction is quite low (Stalikas 2007).
about various modern extraction methods with their Microwave-assisted extraction is another advanced
advantages and disadvantages. method of extraction which has become popular for the
Pressurized fluid extraction has been widely applied for separation of bioactive compounds from plant matrices.
the extraction of flavonoids in recent years (Chen et al. The next section of this paper describes the microwave-
2007d; Husni et al. 2009; Qian et al. 2009; Vichapong et al. assisted extraction process in brief and compares the
2010). This is a controlled extraction method where process with other methods for the extraction in particular
relatively higher temperature and pressure are maintained, of flavonoids from biological samples.
414 Food Bioprocess Technol (2012) 5:409–424

Microwave-Assisted Extraction are used for microwave power applications. The waveguide
can itself be used as the applicator for microwave heating,
Microwaves are non-ionising electromagnetic (EM) waves when the material to be heated is introduced by wall slots
located between the radio-frequency range at the lower and the waveguide is terminated by a matched load. This
frequency and infrared at the higher frequency in the configuration is called a travelling wave device since the
electromagnetic spectrum, within the frequency band of locations of the field maxima change with time. More
300 MHz to 300 GHz; 915 MHz is considered most useful common in the food industry and domestic oven applica-
for industrial applications with its greater penetration depth, tions are standing wave devices, where the microwaves
while 2,450 MHz frequency is generally used in domestic irradiate by slot arrays (that cut the wall currents) or horn
microwave ovens and for extraction applications with a antennas (specially formed open ends) of waveguides
wide range of commercial units designed for analytical (Regier and Schubert 2005).
chemistry purposes. Microwave ovens can be monomode or multimode
The distribution of EM energy in microwave-assisted cavities. The monomode cavity (used in case of a
systems is governed by Maxwell’s equations with appro- focussed microwave oven) can generate a frequency
priate boundary conditions defined by the configuration of which excites only one mode of resonance. The sample
the systems and the interfaces between the treated materials can be placed at the maximum of the electrical field as
and remaining cavity space. The dielectric properties of the the distribution of the field is known. The multimode
samples are the main property parameters of the Maxwell cavity is large and the incident wave is able to affect
equations and, as such, significantly influence the efficiency several modes of resonance. This superimposition of
of EM energy coupled to the materials, EM field distribution modes allows the homogenisation of the field. Systems
and conversion of EM energy into thermal energy within like rotating plates are included for homogenisation.
those biological samples. The dielectric properties of a Microwave extraction can be operated under pressure
material are described by the complex relative permittivity (with or without regulation) or at atmospheric pressure
(ε* relative to that of free space) in the following (Letellier and Budzinski 1999). Use of microwave
relationship: domestic ovens in the laboratory was mentioned for the
p first time by Abu-Samra et al., in 1975 for the treatment of
"» ¼ "0  j"00 ; where j ¼ ð1Þ: biological samples for metal analysis. The earliest studies
regarding microwave extraction were by Ganzler et al.
The real part ε′, is the dielectric constant that reflects the (1986). They used a microwave oven to heat biological
ability of the material to store electric energy when in an samples and soil sample to extract organic compounds.
EM field; the imaginary part ε′′, is the dielectric loss factor Paré et al. (1991), patented a process for the microwave
that influences the conversion of the EM energy into extraction of natural products in 1991. Presently, two
thermal energy. The ratio of the real and imaginary parts of types of commercially available microwave heating
permittivity represents another important parameter, the extraction systems have been developed: the closed-
tangent of loss angle (tan δe =ε′′/ε′), which along with the vessel system and the open-vessel system. The former is
dielectric constant determines the attenuation of microwave usually called microwave-assisted extraction (MAE), by
power in a biological matrix. When exposed to an EM field, which many compounds with bioactivity can be extracted.
the amount of energy converted to heat in biological The latter is named focused microwave-assisted soxhlet or
material is proportional to the value of the loss factor ε′′ solvent extraction, in which the sample is immersed in a
(Datta et al. 2005; Tang 2005). microwave-absorbing solvent in an open vessel and
Each microwave system consists normally of three basic irradiated with focused microwave energy till the comple-
parts: the microwave source, the wave guide and the tion of extraction.
applicator. The microwave source is a magnetron. A The main matrix of extraction in case of flavonoids is the
magnetron consists of a vacuum tube with a central biological matrix and it is well known that the quality of an
electron-emitting cathode of highly negative potential extraction from a plant material strongly depends on the
which is surrounded by a structured anode that forms degree of isolation of the analytes from their matrix. The
cavities, which are coupled by the fringing fields and have lower the level of an analyte in a matrix, the more essential
the intended microwave resonant frequency. The power is its recovery and the more important is the development
output of a magnetron can be controlled by the tube current of effective isolation procedures for accurate analysis
or the magnetic field strength. For guiding an electromag- (Wianowska et al. 2009). In the case of flavonoids, this
netic wave, transmission lines and wave guides can be rule fully applies. With the increasing energy prices and
used. Owing to the lower power losses of waveguides at environmental target to reduce CO2 emissions, there is a
higher frequencies such as those of microwaves, these parts drive to use new techniques for the extraction of natural
Food Bioprocess Technol (2012) 5:409–424 415

substances, which typically use less solvent and energy. In order to better understand the process mechanisms,
The specific features of the interaction between micro- the next section will detail the factors which affect
wave radiation and solvents are quite supportive in this microwave extraction.
regard, hence this method is being increasingly used and
allows significant enhancement of the efficiency of
extraction of organic components (Kubrakova and Factors Affecting Microwave Extraction
Toropchenova 2008). With microwave heating, the energy
transfer occurs via two mechanisms, specifically, by Effect of Solvent System
dipole rotation and ionic conduction, i.e., by means of
reversals of dipoles and displacement of charged ions Solvent is one of the most important factors in an extraction
present in the solute as well as the solvent. The radiation process, particularly more so in a microwave environment.
frequency corresponds to the rotational motion of mole- In general, the higher the dielectric constant and dielectric
cules; in condensed matter, energy absorption immediately loss, the higher is the capacity of the solvent to absorb
causes energy redistribution between molecules and microwave energy, which can lead to faster rate of heating
homogeneous heating of the medium (Kubrakova and of the solvent with respect to the plant material. But the
Toropchenova 2008). During microwave heating, the solvent properties can be modified by combining different
disruption of weak hydrogen bonds promoted by the solvents, which also leads to varying selectivity of the
dipole rotation of the molecules takes place with related solvent for different compounds. For extraction of thermo-
heat release. Also there is migration of dissolved ions labile compounds, a solvent combination with relatively
which increases penetration of the solvent into the matrix, lower dielectric properties can be used to ensure that the
thus, facilitating the collection of the target compounds solvent temperature will remain lower to cool-off the
(Mandal et al. 2007). To extract any complex constituent solutes once they are liberated into the solvent. In this
from a biological source, first it has to be freed from the case, the microwave energy interacts preferentially with the
matrix with cellular release into a proper solvent (Jain et plant matrix (Kaufmann and Christen 2002) leading to
al. 2009; Cannell 1998). During microwave processing, a efficient release of the plant compounds in the cooler
considerable amount of pressure builds up inside the solvent. Extraction efficiency depends on the solubility of
biomaterial which modifies the physical properties of the the analytes in the extraction solvent. Both polar (Li et al.
biological tissues improving the porosity of the biological 2009; Casazza et al. 2010) and non-polar solvents
matrix, which allows better penetration of extracting (Barnabas et al. 1995) can be used in microwave extraction.
solvent through the matrix (Kratchanova et al. 2004; Yeoh Flavonoids vary in polarity, so the solvents can be highly
et al. 2008). The pressure building effect of the microwave polar or less polar. Less polar solvents are used for
leads to swelling which generally forces the cells to split. extraction of flavonoid aglycones and more polar solvents
In the case of extraction of pectin from orange peel, it has are used for extraction of flavonoid glycosides and
been observed that the degree of disintegration with anthocyanin. Benzene, chloroform, ether, and ethyl acetate
microwave extraction was much more as compared with are the common solvents for the extraction of less polar
the traditional method (Zhongdong et al. 2006). Whereas aglycones such as isoflavones, flavanones, dihydroflavo-
in the case of traditional extraction method the pectin nols, flavones, and flavonols which are highly methylated
mainly remained in cluster formation, while in case of (Harborne et al. 1975). The more polar flavonoid glyco-
microwave extraction the cluster formation was broken sides such as hydroxylated flavones, flavonols, biflavonyls,
and pectin became particulate in a way that was similar to aurones, and chalcones are traditionally extracted using
crystals (Zhongdong et al. 2006). Sometimes, the process acetone, alcohol, or water (Harborne et al. 1975). For
of microwave extraction can be enhanced by a mechanical microwave extraction of both polar aglycones and flavo-
shearing module which uses physical force to fracture the noid glycosides, the solvents commonly used include
plant cell walls which can further speed up the dissolution methanol or ethanol, with different concentrations obtained
of the cells active ingredients into the solution (Jang et al. by dilution with water (Ding et al. 2003; Terigar et al. 2010;
2009). There are many cases when microwave is either Tsukayama et al. 2010). Also solvents such as acetone
directly used for the extraction (with or without using (Kothari and Seshadri 2010) and chloroform are used for
solvents) or used for pretreatment before carrying out the microwave-assisted extraction. The variation in the concen-
final processing which leads to an increase in yield of the tration of solvents also affects the flavonoid extraction as
final product. For example, microwave pretreatment used observed in the case of microwave extraction of quercetin
for heating the apple mash before pressing the juice lead and rutin using different concentrations of ethanol (Zhang
to an increase of flavonoid content in the juice (Roberts et al. 2009). Extraction yield was found to increase when
and Gerard 2004). the ethanol concentration was increased from 30% to 50%
416 Food Bioprocess Technol (2012) 5:409–424

and decrease with the further dilution of ethanol (Zhang et extraction yield, preventing prolonged heating using the
al. 2009). There are many other examples supporting the same volume of solvent. Use of fresh batches of solvent
fact that the extraction efficiency is affected by the type of prevents the saturation of the solvent with solute, increasing
solvent used for extraction (Kiss et al. 2000; Escribano- the mass transfer and extraction kinetics.
Bailon and Santos-Buelga 2003; Song et al. 2007) along
with its different dilution levels obtained using water (Chen Effect of Microwave Power Level
et al. 2008; Song et al. 2007). It was observed that not only
the composition of the solvent system but also the pH of the The intensity of the incident microwave power controls the
solvent can significantly affect the different efficiency of amount of energy supplied to the sample which is
the extraction steps (Stalikas 2007). As scientists are more converted to heat energy in the dielectric material to
concerned about environmental impacts, the solvent vol- increase its temperature. It affects interactions and equilib-
ume becomes another important factor to be considered and rium rate and controls partition of analytes between sample
usually with microwave extraction, the total consumption and solvent (Ma et al. 2009). Generally, it has been
volume of solvent decreases (Chen et al. 2007a). However, observed that there is an increase in the yield of the
it has also been reported that in the case of a closed-vessel extracted compound when there is an increase in the
microwave extraction system, the heating is faster when a microwave power (Hu et al. 2008). A study revealed that
larger volume of solvent is used (Letellier and Budzinski when microwave power was increased from 200 to
1999). 1,000 W, the extraction yield of flavonoid from Radix
Ionic liquid solutions can also be used as potential astragali was increased. However, high microwave power
solvents for microwave-assisted extraction of polyphenolic might increase the product temperature overly high, and
compounds, including flavonoids such as quercetin, which decrease the extraction yield through product damage or
was extracted by Du et al. (2009) from medicinal plants compound breakdown (Ma et al. 2009). Also, a decrease in
samples of Psidium guajava Linn. leaves and Smilax china the flavonoid yields with increase in power has been found
tubers. Ionic liquids, their concentration and the to be significant when the extraction period was extended
microwave-assisted extraction conditions were investigated. (Gao et al. 2006; Xiao et al. 2008).
The anions and cations of ionic liquids had influences on
the extraction of polyphenolic compounds and the ionic Effect of Temperature
liquids with electron-rich aromatic π-system were found to
enhance the extraction yield. High-temperature extraction can be profitable with the
resulting increase in solubility. This is because higher
Effect of Time of Application of Microwave temperature causes increased intermolecular interactions
within the solvent, giving rise to higher molecular motion
In many of the reported studies, it has been observed that which increases the solubility. The increasing temperature
the extraction yield tends to increase with an increase of the may also cause a cellular pressure build up which may
extraction time, but the increase in yield was found to be cause cell rupture and opening of the cell matrix, and as a
very small with further increase in time (Wang et al. result, increased components’ availability to be extracted
2008b). In the case of flavonoids extracted from Radix into the solution. Moreover, at high temperature, the solvent
astragali, there was an increase in yield with time up to an viscosity decreases, increasing its mobility and solubility,
exposure of 25 min and then the extraction yield started thus increasing the efficiency of extraction (Khajeh et al.
decreasing (Xiao et al. 2008). As the rate of heating of the 2009). However, in some cases, it has been observed that
polar solvents such as ethanol and methanol is quite high, the extraction efficiency increases with the increase in
because of the comparatively high dielectric properties, and temperature until an optimum temperature and solvent to
further dilution with water increases the heating capacity of solid ratio is obtained and then it starts decreasing with the
the solvent combination, prolonged application of micro- further increase in temperature. This also varies with the
wave may lead to the degradation of the target compounds type of compounds to be extracted as the degradation
with overheating of the solute/solvent system. Nonetheless, temperature of every compound is different. Higher
the extraction time has been found generally to have a temperature or prolonged application of high temperature,
positive influence on the yield of the extracted product induced by prolonged application of high power levels can
(Lucchesi et al. 2007). Sometimes the samples are extracted lead to degradation and subsequent decrease in yield and
in multiple steps using, consecutive extraction cycles, quality.
which is also an example of use of greater volume of Liazid et al. (2007) investigated the stability of phenolic
solvent and higher microwave application time (Xiao et al. compounds during microwave extraction. It was observed
2008). This practice helps in further improving the that out of the 22 compounds of different phenolic families
Food Bioprocess Technol (2012) 5:409–424 417

(benzoic acids, benzoic aldehydes, cinnamic acids, cate- applications and sample extraction. Examples of successful
chins, coumarins, stilbens, and flavonols), all the com- applications of MAE for extraction of flavonoids in recent
pounds studied were stable at 100 °C, whereas at 125 °C years are mentioned in Table 2.
there was significant degradation of epicatechin, resvera- As compared with conventional methods of extraction,
trol, and myricetin. It was concluded that compounds which microwave extraction can take much less time. For
have a greater number of hydroxyl-type substituents, are example, soxhlet extraction takes at least 1 h and
more easily degraded under high-temperature extraction sometimes 12–24 h for most extractions and consumes a
conditions. The working temperature during their study was lot of solvent, microwave extraction takes around 30 min or
varied from 50 to 175 °C (Liazid et al. 2007). less, thus significantly decreasing the time required for
Similar observations were made by Xiao et al. (2008), extraction, which is a very important factor in case of
where the yield of flavonoids increased remarkably with the analytical chemistry applications (Chen et al. 2007a; Ding
increase of temperature from 90 to 110 °C while above et al. 2003; Gao et al. 2006; Kothari and Seshadri 2010).
110 °C, the yield increased only slowly and the extract was Prolonged extraction periods and high-temperature
found to be scorched. exposures in the case of soxhlet can affect significantly
the quality of the extract. Thermolabile compounds are lost
Effect of Contact Surface Area during the process which leads to lower yields. But in case
of microwave extraction, as the process is faster, the yield
When the contact surface area of the plant material of the bioactive compounds is generally higher and
increases, then the extraction efficiency generally increases. comparable to modern extraction methods such as pressur-
Because of this, the sample preparation steps include the ized liquid extraction, supercritical liquid extraction and
steps of milling, grinding and homogenisation so that the subcritical extraction.
interaction of the biological cellular matrix with the solvent Another drawback of conventional extraction is that it
increases. This concept is also applied for microwave influences the integrity of flavonoid glycosides during
extraction and for flavonoids extraction. Xiao et al. (2008) prolonged extraction thus affecting the reproducibility and
blended and passed the blended sample through a sieve stability (Stalikas 2007), whereas the degradation of the
before drying to obtain a uniform contact surface sample. flavonoids is prevented during microwave extraction (Gao
Kothari and Seshadri (2010) used finely ground seeds for et al. 2006; Xiao et al. 2008). For example, during the
the extraction of flavonoids from Annona squamosa and conventional extraction of isoflavonoids and saponins from
Carica papaya seeds. Radix astragali, it was observed that flavonoid glycoside
malonates were converted into their related glycosides
during the prolonged conventional extraction, thus affecting
Application and Benefits of MAE for Extraction the reproducibility. However, the glycosidic conversion was
of Flavonoids inhibited when using microwave-assisted extraction (Song
et al. 2007). Increase in reproducibility of the analytical
Microwave-assisted extraction has several advantages, procedures leads to better results in analytical research and
which have been well established since first reported by compound quantification applications. Generally, the anal-
Ganzler et al., in 1986 and the technology has evolved well ysis regarding the stability of the flavonoids (Xiao et al.
with time, with developed systems for analytical chemistry 2008) is carried out before extraction which helps the

Table 2 Various examples of application of microwave-assisted extraction for extraction of flavonoids in recent years

Biological material Solvent Reference

Epimedium koreamum Nakai Ethanol (40%) Liu et al. 2006

Achillea millefolium Proa Methanol Trendafilova and Todorova 2008
Radix astragali Ethanol (90%) Xiao et al. 2008
Morus alba L. leaves Ethanol (60%) Li et al. 2009
Alpinia zerumbet (Pers.) Burtt et Smith leaves Ethanol (70%) and water Victório et al. 2009
Vitis vinifera waste Methanol Casazza et al. 2010
Buddleia officinalis Maxim Ethanol Pan et al. 2010
Radix puerariae Ethanol (70%) Wang et al. 2010
Portulaca oleracea L. Ethanol (70% v/v) Zhu et al. 2010
Uncaria sinensis Ultra pure water Tan et al. 2011
418 Food Bioprocess Technol (2012) 5:409–424

researchers to know the temperature of degradation. This cultured cells and in other cases such as the extraction of
helps in establishing the safe temperature of extraction and flavonoids from Flos sophaoae (Li et al. 2004). Dynamic
proper optimisation of the process (Mao et al. 2008; Yan et microwave-assisted extraction can be coupled with an
al. 2010). analytical step directly as done in the determination of
Over the years, modifications and optimization of andrographolide and dehydroandrographolide in Androgra-
established microwave extraction methodologies have phis paniculata Nees using high-performance liquid chro-
resulted in better outputs in terms of prevention of the matography (Chen et al. 2007c). There are other similar
degradation of flavonoids. Some of the advanced methods applications in different cases (Chen et al. 2007b; Chen et
of extraction using microwave have been summarized in al. 2006), but there is comparatively fewer report of
Table 3. An apparatus for extraction of flavonoids from application of this technology with flavonoids (Chen et al.
Herba epimedii was constructed which utilized a micro- 2007a).
wave technique for heating in a dynamic mode (Chen et al. Focussed microwave soxhlet extraction of compounds can
2008). A laboratory TM010 microwave resonance cavity prove to be an efficient combination of soxhlet and microwave
built by Chen et al. (2008) was used to concentrate the extraction and has been reported by many (Garcia-Ayuso et al.
microwave energy and thus the power needed to achieve 1998; Luque de Castro and Garcia-Ayuso 1998; Prados-
efficient extraction was significantly reduced leading to Rosales et al. 2002 and 2003), but its application for
energy savings. When the method was compared with other flavonoids from biological matrices is still missing from the
extraction methods such as pressurized microwave-assisted literature. As the process seems to be promising in other
extraction, ultrasonic extraction, heat reflux extraction and fields, it can be expected that application of this new
soxhlet extraction, the results showed that the extraction technology can be helpful with flavonoids as well.
yield of flavonoids in Herba epimedii obtained by dynamic Another remarkable advantage of microwave use is that
microwave-assisted extraction (DMAE), where there is both polar and non-polar compounds can be simultaneously
continuous flow of fresh solvent through the microwave extracted during microwave extraction. Simultaneous MAE
extractor, was higher than that obtained by other methods. of polar (biophenols) and non-polar (fatty acids) com-
The extraction yield obtained by DMAE was found to be pounds from alperujo (a residue obtained from the two-
similar to that obtained by pressurized microwave-assisted phase decantation process used in the olive oil industry)
extraction. But the flavonoids in Herba epimedii did not was applied successfully using a mixture of two suitable
decompose easily when increasing the extraction time in extraction solvents in a short process time (Pérez-Serradilla
DMAE. The benefit of dynamic extraction system is that it et al. 2007). Prevention of extra steps consisting of
can transfer the analytes extracted from the sample matrix application of individual processes for the extraction of
with the fresh-flowing solvent out of the extraction vessel polar and non-polar compounds helps in preventing
preventing the damage to the compounds at higher degradation of the biological matrix and the target
operating temperatures (Chen et al. 2008). A similar type compound.
of technology was also used by Gao et al. (2006) for Use of polar solvents helps in efficient microwave
extraction of flavonoids from Saussurea medusa Maxim heating of the solvent, which leads to efficient heating of

Table 3 Advanced methods of microwave extraction for extraction of flavonoids used in recent years

Method Biomaterial Reference

High-pressure microwave-assisted extraction Acanthopanax senticosus leaves Liu et al. 2005

Dynamic microwave-assisted extraction coupled Platycladus orientalis (L.) Franco Chen et al. 2007a
with on-line derivatization and ultraviolet–visible
detection
Dynamic microwave-assisted extraction directly Andrographis paniculata nees Chen et al. 2007c
coupled with analytical step high-performance
liquid chromatography
Dynamic microwave-assisted extraction Herba epimedii Chen et al. 2008
Vacuum microwave-assisted extraction Chinese herbs (Rhizoma Polygoni Wang et al. 2008a
Cuspidati, Myrica rubra, and Flos
carthami)
Solvent-free microwave hydro-diffusion Onion (Allium cepa L.) Zill-e-Huma et al. 2009
and gravity extraction
Solvent-free microwave hydro-diffusion Sea buckthorn (Hippophae rhamnoides) food by-products Périno-Issartier et al. 2010
and gravity technique
Food Bioprocess Technol (2012) 5:409–424 419

the solvent/biological matrix and the more rapid extraction Comparison of Microwave-Assisted Extraction
of the flavonoids. Also solvents like hexane and chloro- with Other Modern Extraction Methods
form, which are more transparent to the EM (absorbing less
energy), can be used effectively to extract thermolabile In analytical chemistry applications, it has been observed
compounds. In the case of flavonoids, generally polar that microwave extraction obtains similar or greater yields
solvents are used although ionic solvents have also been than ultrasonic extraction in the case of flavonoids (Song et
used. Water can also be a potential solvent for both polar al. 2007) and combining both has been observed to increase
and non-polar compounds when using microwave for the extraction yield further (Lou et al. 2010). For ultrasound
extraction, which can be an option when a more environ- extraction, maximum power is observed in the vicinity of
mental friendly process is required. As ionic conduction is the radiating surface of the ultrasound horn and as we move
one of the direct effects of microwave heating, the heating further from the source the intensity decreases. The
rate increases with the addition of ionic solvents (Sparr presence of solid particles may decrease the intensity
Eskilsson and Björklund 2000). Ionic solvents can absorb further. Hence, additional agitation and shaking is generally
microwave energy efficiently and increase the microwave necessary to avoid standing waves or formation of solid
heating selectivity also (Du et al. 2007; Du et al. 2009). free regions to ensure adequate stimulation for enhanced
Solvent-free microwave extraction is the recent develop- extraction (Wang and Weller 2006). While for microwave
ment in the microwave extraction field and has been widely extraction, the optimal process takes place in the region of
used for essential oil extraction (Lucchesi et al. 2004a; maximum electromagnetic power which may also require
Lucchesi et al. 2004b; Lucchesi et al. 2007; Wang et al. agitation to improve the performance. Hence, the combina-
2006) but its use for flavonoids has yet to be explored. tion of both methods can alleviate the burden of their
Also it is reported that one of the greatest advantages of weaknesses to enhance extraction.
the MAE is the lower use of organic solvents, which can be Closed-vessel microwave-assisted extraction has been
further enhanced by the reuse of the organic solvents by use described to be similar to pressurized liquid extraction
of a condenser with continuous flow of freezing water to which is successfully used for flavonoid extraction (Chen et
condense the escaping vapour, so that the solvent can be al. 2007d; Qian et al. 2009). Closed systems allow
recycled into the extraction process repeatedly (Mao et al. conducting experiments in a closed environment which
2007). prevents contamination and loss of volatile compounds and
There is a definite advantage of using microwave- they reduce the possible errors during transfer of sample
assisted extraction for small samples as the technology from one container to another. Closed-vessel microwave
allows the determination of analytes in small samples as extraction is generally used for extreme conditions of
low as 0.1 g. Small sample sizes help in maintaining the temperature and pressure which may not be best suited for
compound concentrations in extracts to overcome the loss food applications (Llompart et al. 1997a; Llompart et al.
of sensitivity caused by dilution (Jin et al. 1999). Extraction 1997b). Use of a closed-vessel microwave system under
with low volume of solvents decreases the time required for strict and controlled settings for the extraction of flavonoids
the evaporation of these solvents which could be the next should be explored, as it could produce better outputs
step of improvements in analytical research. This also similar to or better than pressurised liquid extraction,
prevents the degradation of the compounds during the reducing further the time and microwave energy required.
prolonged heating required for the evaporation of the As mentioned earlier, supercritical fluid extraction is a
solvent. promising method in terms of extraction yield and it is one
The conventional extraction of flavanoids often involves of the methods most comparable with microwave-assisted
the addition of an acid. Using microwave extraction can, in extraction. However, some of the operating attributes of
most of the cases, reduce the need for an acid (Gao et al. microwave extraction are more versatile than in the case of
2006; Kothari and Seshadri 2010; Xiao et al. 2008). supercritical fluid extraction. In supercritical extraction,
However, in cases where acids are used, such as for the CO2 is the major solvent, but the process is not efficient for
extraction of anthocyanins (Sun et al. 2007; Yang and Zhai the extraction of polar compounds. For the extraction of
2010), less concentrated acids can be used with microwave polar compounds, polar solvents such as methanol or
as compared with conventional methods of extraction, ethanol have to be added which decreases the extraction
without affecting the final yield. Use of less concentrated selectivity. The high concentration of a polarity modifier
acids reduces the safety hazards caused by fast and may lead to a mixture temperature lower than the critical
excessive pressure build up in the reaction vessels. The temperature, which cancels the benefit of using supercritical
oxidizing power of the dilute acids might be lower but they extraction (Camel 2001). On the other hand, microwave
absorb more microwave energy as there is more water extraction can be used for the separation of both polar and
(Wong et al. 1997). non-polar compounds efficiently. A major advantage
420 Food Bioprocess Technol (2012) 5:409–424

remains in the case of supercritical fluid extraction for its lites) can then be collected and separated in receiving
suitability in the extraction of volatile compounds. This flasks. It is important to note that this method allows the
advantageous property can be obtained with microwave extraction of natural substances without distillation or
extraction by using a closed system and cooling the solvent extraction which are the most energy and solvent
extraction vessel to lower temperature before pressure consuming unit operations. Losses of some volatile com-
release (Kaufmann and Christen 2002). Another advantage pounds, long extraction times, degradation of unsaturated
of using microwave extraction rather than supercritical fluid or ester compounds through thermal or hydrolytic effects
extraction is that drying of the samples can be avoided for are some other principal disadvantages of the common
sample preparation with microwave extraction, whereas all extraction methods such as traditional hydro-distillation or
samples are usually dried before supercritical extraction steam distillation. These disadvantages are overcome in
(Stalikas 2007). When microwave is used for extraction, solvent-free microwave extraction, which is becoming more
water is a key parameter since microwaves interact with the and more popular for the extraction of phytochemicals.
water molecules present in the cellular glands and vascular Microwave-assisted extraction has been reported to be
systems of the plant’s biological matrix, leading to rapid an economical substitute for routinely used homogenization
heating and temperature increase. This subsequently leads and vortexing extraction techniques (Akhtar 2005). Also it
to rupture of cell walls and fast release of bioactive has been observed that the MAE method followed by
compounds (Camel 2001). Supercritical fluid extraction is HPLC-UV-ELSD determination is a simple, rapid, and
also more matrix dependent as for each matrix the process reliable method for the quality assessment of the extracts
of extraction of certain compound has to be optimized. (Song et al. 2007). Microwave steam diffusion is one of the
Microwave extraction is less matrix dependent as it initially other methods which was developed as a cleaner and new
uses less selective conditions in using wide spectrum process designed and operated for isolation of essential oils
organic solvents (Camel 2001). and was compared with conventional steam diffusion.
Microwave extraction is a newer method with its own Combination of MAE with the solid-phase microwave
benefits and drawbacks, nonetheless with the many obser- extraction has also been tried as a method of extraction of
vations and research from literature, microwave-assisted volatile compounds in some of the plant materials and
extraction seems to have continued potential applications. during these studies the yield of the final product has been
found to be high and less time consuming (Zhou et al.
2009). This shows great promise for microwave extraction
Future of MAE of Flavonoids of flavonoids.
Sometimes the microwave-assisted extraction process is
Microwave-assisted extraction has remarkable outputs of supported by high pressure which significantly enhances
yield, quality, and selectivity which have helped in the efficiency of the extraction process. In high-pressure
retaining its utility. With continued modification of equip- microwave-assisted extraction, the rate of system tempera-
ment and use of microwave, developments have contributed ture and pressure increase seemed to be related to the
in establishing it as one of the leading analytical extraction solvent’s ability to absorb microwave energy, its specific
methods. Over the years there have been some major heat and its heat of vaporization. The combination of
modifications in the basic extraction processes using microwave technology and pressure has been successfully
microwave, and they are briefly mentioned below. applied for the extraction of ginsenosides (Zhang et al.
An extraction process known as microwave hydro- 2008; Wang et al. 2008b), and shows potential for the
diffusion was developed using the combination of micro- extraction of flavonoids.
wave heating and gravity working at atmospheric pressure. It has also been shown that the coupling of microwave-
In this simple method, the plant material is directly placed assisted extraction and cloud-point extraction (extraction
in a microwave reactor without any added solvent or water. using phase separation at a temperature, specific for a
The internal heating of the in situ water within the plant solvent at which dissolved solids are no longer completely
material distends the plant cells and leads to rupture of the soluble) could be employed as a new and effective
glands and cell receptacles. Heating under microwaves thus approach for the rapid extraction and pre-concentration of
liberates molecules of interest together with in situ water. pharmacologically active ingredients from liquorice root
This physical phenomenon, known as hydro-diffusion, without disturbing the subsequent chromatographic analysis
allows the extract to diffuse outside the plant material and (Sun et al. 2008). The mass of the analytical portion must
drop by gravity out of the microwave reactor through a be carefully selected to prevent excessive pressure during
perforated disc (Ferhat et al. 2007). A heat exchanger the digestion process. For each food matrix, the optimum
outside the microwave oven then continuously cools the microwave conditions are governed by their properties
extract. Crude juice or extract (in situ water and metabo- (Khajeh et al. 2009).
Food Bioprocess Technol (2012) 5:409–424 421

Conclusions Chandra, A. K., De, N., & Roy Choudhury, S. (2010). Effect of
different doses of un-fractionated green & black tea extracts on
thyroid physiology. Human & Experimental Toxicology, doi:
In general, microwave-assisted extractions processes are either 10.1177/0960327110382563.
better or comparable with conventional solvent extraction Chen, L., Ding, L., Zhang, H., Li, J., Wang, Y., Wang, X., et al.
methods. Optimisation of process parameters and the use of (2006). Dynamic microwave-assisted extraction coupled with on-
line spectrophotometric determination of safflower yellow in
modern technologies can significantly improve extraction
Flos carthami. Analytica Chimica Acta, 580(1), 75–82.
efficiencies. Hence, for the improvement of the extraction of Chen, L. G., Ding, L., Yu, A. M., Yang, R. L., Wang, X. P., Li, J. T., et al.
flavonoids, there is still a lot to explore in terms of extraction (2007a). Continuous determination of total flavonoids in Platycla-
process parameters and technologies. There has been much dus orientalis (L.) Franco by dynamic microwave-assisted extrac-
tion coupled with on-line derivatization and ultraviolet-visible
research and development advances in the microwave-assisted detection. Analytica Chimica Acta, 596(1), 164–170.
extraction of a number of plant compounds, however, very little Chen, L., Ding, L., Jin, H., Song, D., Zhang, H., Li, J., et al. (2007b).
progress has been made for the microwave-assisted extraction The determination of organochlorine pesticides based on dynam-
of flavonoids. By taking into account all the process factors and ic microwave-assisted extraction coupled with on-line solid-
phase extraction of high-performance liquid chromatography.
their potential combination, an optimized process can be
Analytica Chimica Acta, 589(2), 239–246.
developed. Analytical chemistry is a vast field and it seems Chen, L., Jin, H., Ding, L., Zhang, H., Wang, X., Wang, Z., et al.
microwave will retain its position as both an established and a (2007c). On-line coupling of dynamic microwave-assisted ex-
promising process with its benefits and application develop- traction with high-performance liquid chromatography for deter-
mination of andrographolide and dehydroandrographolide in
ment potential from modified technological components.
Andrographis paniculata Nees. Journal of Chromatography A,
1140(1–2), 71–77.
Chen, X. J., Guo, B. L., Li, S. P., Zhang, Q. W., Tu, P. F., & Wang, Y.
T. (2007d). Simultaneous determination of 15 flavonoids in
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