CHAPTER TEN

Optimization of extraction
methodologies and purification
technologies to recover
phytonutrients from food
Tarun Belwal*, Hari Prasad Devkota†,‡, Sudipta Ramola§,
Harish Chandra Andola¶, Indra D. Bhatt*
*Centre for Biodiversity Conservation and Management, G. B. Pant National Institute of Himalayan
Environment and Sustainable Development, Kosi-Katarmal, Almora, India
†
School of Pharmacy, Kumamoto University, Kumamoto, Japan
‡
Program for Leading Graduate Schools, Health Life Science: Interdisciplinary and Glocal Oriented (HIGO)
Program, Kumamoto University, Kumamoto, Japan
§
DBS School of Agriculture and Allied Sciences, Dehradun, India
¶
School of Environment and Natural Resources (SENR), Doon University, Dehradun, India

10.1 Introduction
Phytonutrients are classified into various classes based on their chem-
ical structure and functional attributes. These commonly includes polyphe-
nols (phenolic acids, flavonoids, tannins, lignins anthocyanins), alkaloids,
steroids, carotenoids, terpenoids, and so forth (Chadwick and Marsh,
2008). Phytonutrients are commonly known as phytochemicals, or second-
ary metabolites, which play an important role during stress, and act as the
defensive system of plants. Phytonutrients are proven to fight against oxida-
tive stress, fungal infection, or many other kinds of external stimuli (Agrawal
and Weber, 2015). They are also tested on humans and animal models, and
have shown their effectiveness against various diseases (Srinivasan et al.,
2001). Different processes have been followed to make phytonutrients
available as extracts and/or in their pure form, for various pharmaceutical,
cosmeceutical, and nutraceutical products formulation. These include pro-
cesses such as harvesting of plant materials, washing, drying (optional),
extraction of phytonutrients, purification of compound of interest, testing
in animal models against several diseases, clinical trials, and finally, the
phytonutrient product formulation (Fig. 10.1). These processes depend
upon various factors, which can be optimized for better outcomes
(Fig. 10.1).

Phytonutrients in Food Copyright © 2020 Elsevier Inc. 217

https://doi.org/10.1016/B978-0-12-815354-3.00007-1 All rights reserved.
218 Tarun Belwal et al.

Factors affecting phytonutrient Optimization strategy

concentration
Collection of raw Seasons, location, climatic condition, Only possible in artificial
materials plant growth stage systems

Drying Drying conditions (temperature, time,

etc.)

Extraction conditions (type, temperature,

Extraction time, solvent type, concentration, power, Optimization could be
frequency, particle size, etc.) possible

Extraction conditions (mobile phase, flow

Purification rate, column temperature, column type,
chromatography type, etc.)

Phytonutrient
of interest

Solubility, miscibility, bioavailability, Optimization could be

Product formulations possible
compatibility, effectiveness, toxicity, etc.

Fig. 10.1 Phytonutrients processing steps and its factors and optimization strategy.

Extraction and purification of phytonutrients are the most crucial and

important processing steps during phytonutrient product formulation
(Belwal et al., 2017a). Extraction involves extracting the compounds from
the plant matrix without affecting its physicochemical properties. For the
extraction process, various solvents are used, depending on the compound
of interest and solvent toxicity. These solvents disrupt the plant cell and dis-
solve the phytonutrients. Various approaches such as heating, sonication,
high pressure, and so forth are also applied during the process of extraction
(Belwal et al., 2018). Traditionally, phytonutrient extraction is done by var-
ious conventional methods that use prolonged heating, such as percolation,
maceration, and processes, such as Soxhlet extraction. With changing
requirements and industrial needs, the use of various modern and sophisti-
cated techniques of extraction such as microwave, sonication, supercritical
fluids, high pressure, and so forth have been utilized (Belwal et al., 2018).
These processes have advantages of using a lower volume of solvent, or
no solvent, and take less extraction time, which results in a higher yield
of compounds without affecting its physicochemical properties, and so
are also categorized as green extraction techniques (Chemat et al., 2012).
Extraction results in the end-product in the form of an extract consisting
Optimization of extraction methodologies 219

of a mixture of phytonutrients. These phytonutrients need to be identified in

terms of their quantity and quality, and must be purified in their pure form to
be tested against various diseases or utilized in various product formulation.
The purification process includes various methods, such as chromatography,
crystallography, partition techniques, and so forth. Various optimization
strategies (single factor and multiple factors) are adopted in order to maxi-
mize the extraction and purification efficiency, which defines the correct
combination of different process factors and their level to maximize the
responses with respect to phytonutrient content and concentration.
It has been found that these optimization strategies have been successfully
examined for obtaining higher quantity and quality of phytonutrients from
various plant materials (Gan and Latiff, 2011; Belwal et al., 2016, 2017a,b).
This chapter includes different techniques of phytonutrient extraction and
purification, and strategies for optimization of the same.

10.2 Extraction and purification techniques for

phytonutrients
Extraction of plant materials is the first step for obtaining bioactive
phytonutrients from plant materials. Selection of proper extraction solvent,
and conditions such as temperature, time of exposure, solid liquid ratio, and
so forth are important for extracting and separating phytonutrients from the
complex mixture of compounds. Conventional methods of extraction such
as maceration, infusion, decoction, and percolation have been in use for a
very long time. Significant progress has been made in the past decade regard-
ing the design and development of nonconventional or advanced types of
extraction methods, such as microwave assisted extraction (MAE), super-
critical fluid extraction (SFE), ultrasonic assisted extraction (UAE), pressur-
ized liquid extraction (PLE), pulsed electric field assisted extraction (PEF),
and enzyme assisted extraction (EAE). Some of the features of traditional/
conventional and modern/nonconventional extraction methods are
explained as follows.

10.2.1 Traditional/conventional extraction methods

Traditional/conventional extraction methods using water or organic sol-
vents (e.g., ethanol) have been in practice for decades for obtaining desired
phytonutrient rich extracts. Some representative traditional methods of
extraction are given in Fig. 10.2A. Although these methods are economic
and easy to use at any laboratory or company setup, they have many
220 Tarun Belwal et al.

Maceration

Percolation Soxhlet
Extraction
Solvent
Plant sample mixed with
solvent and kept for some
time and filtered

Decoction Plant sample

Soxhlet
apparatus

Water bath
Plant sample mixed with
Plant sample mixed with
solvent is extracted in a
solvent and heated inside the
percolator
Plant sample mixed with apparatus for a specific period
water and boiled till the of time
solvent volume becomes half
and filtered

(A)

Supercritical Fluid Extraction (SFE)

Pump
Mixture
vessel
Pump Pump Pump Pump
Co-solvent
tank

Pump
Separator Separator
Extract collector
Plant sample Soxhlet
apparatus
Extractor

CO2 cylinder

Microwave Assisted Extraction (MAE)

Condenser

Microwave oven

Plant sample
and solvent

(B)
Fig. 10.2 Extraction methods for phytonutrients. (A) Conventional extraction methods
and (B) Advanced extraction methods.
Optimization of extraction methodologies 221

limitations as compared with nonconventional methods of extraction.

Traditional methods require a large amount of sample volume, and are time
consuming. Many of these methods, such as decoction and Soxhlet extrac-
tion, also use high temperature, which may result in the degradation/
decomposition of the desired phytonutrients. The basic features of some
of them are given in Table 10.1.

10.2.2 Modern/nonconventional extraction methods

In order to overcome the limitations of conventional methods of extraction,
many advance and efficient methods of extraction have been developed in
recent years. These techniques have advantages over conventional methods,
such as less extraction time, and solvent volume with a higher extraction
yield (Belwal et al., 2018; Xu et al., 2017; Azmir et al., 2013). These
new methods are also termed “Green Extraction” methods because of their
low energy consumption and use of alternative solvents (Chemat et al.,
2012). Some representations of these extraction techniques are given in
Fig. 10.2B, and their basic features are given in Table 10.1.
MAE of phytonutrients is performed by the use of electromagnetic radi-
ation at the frequency range of 300 MHz to 300 GHz. The continuous
dipole rotation (dielectric heating) and the frictional resistance to the ion
flow generate heat and increase extraction efficiency (Belwal et al., 2018;
Azmir et al., 2013). Any solvent that can absorb microwaves would be a suit-
able candidate for MAE, besides having good extraction efficiency and
avoiding the solvent toxicity. Two types of instrumentation, closed vessel
type and open vessel type, are available for MAE. The extraction time is usu-
ally less than 1 h, and its extraction efficiency can be increased by the proper
choice of solvent and plant sample, and also by controlling the temperature/
power for selective extraction of phytonutrients.
In recent years supercritical fluids, especially carbon dioxide (CO2) was
used for extraction, and commonly known as supercritical fluid extraction
(SFE). This technique involves the extraction of plant materials using super-
critical CO2 in a high-pressure vessel, and separating the extractives with the
help of a solid phase separator. The main advantages of SEF are reduced
solvent volume, high extraction yield, easy separation of the extracts from
solvents, and so forth (Xu et al., 2017). However, the main limitation of
SFE is the low polarity of the solvent (supercritical CO2), which makes
extraction of polar compounds difficult. This limitation can be solved by
the use of a small amount of polar solvents, such as methanol or ethanol,
Table 10.1 Some basic features of extraction techniques (Belwal et al., 2018)
Soxhlet
Parameters Maceration extraction MAE SFE UAE PLE PEF EAE
Solvent used Ethanol, Nonpolar Any solvent Carbon Ethanol, Ethanol, Organic Enzymes
(preferably) methanol, solvents that absorb dioxide methanol, methanol, solvents (cellulase,
water (hexane, microwave (methanol and water water and and water pectinase,
ether, or without or ethanol other hemicellulase)
petroleum any solvent can be used organic
ether) as co- solvents
solvents)
Temperature Room Heat applied Heat applied Above 31°C If required, heat Applied Not applied Applied (water
temperature applied bath)
Duration 2–5 days 2–18 h 1–40 min Up to 1 h Up to 1 h 1–2 h Few seconds 1–12 h
Volume 100 mL to 3 L Up to 1 L Up to few – Up to 250 mL Up to few Up to few Up to few liters
of solvent hundred hundred hundred
mL mL mL
Pressure Not applied Not applied Not applied Applied Not applied Applied Not applied Not applied
Plant part Any plant part Seed and Any plant Any plant Any plant part Any plant Any plant Any plant part
preferred fruits in part part part part
particular
Large scale Available Available Possible Available Available but Possible Possible Not available
application limited
because of the
higher cost
and
nonlinearity
of process
Green Not possible Not possible Possible Possible Possible Possible Possible Possible
extraction
MAE, microwave assisted extraction; UAE, ultrasonic assisted extraction; SFE, supercritical fluid extraction; PEF, pulsed electric field; PLE, pressurized liquid extraction; EAE, enzyme
assisted extraction.
Optimization of extraction methodologies 223

in the extraction system as co-solvents (Azmir et al., 2013). In recent years,

SFE has been widely used in the extraction of lipids, lipid soluble com-
pounds, and essential oils, which are widely used in food/pharmaceutical
and other allied industries.
Ultrasonic assisted extraction (UAE) uses ultrasonic waves from the fre-
quency range of 20 kHz to 100 MHz, for extraction of phytonutrients by
either an ultrasonic bath and/or ultrasonic probe. While moving in a
medium, ultrasound generate cavitation, which involve compression and
expansion cycles (Azmir et al., 2013). The process of cavitation includes
the formation and collapse of bubbles, which results in the formation of high
pressure and temperature and thus helps in the extraction of phytonutrients
from plant matrices (Belwal et al., 2018; Azmir et al., 2013). Advantages of
UAE are low solvent use, fast extraction (usually less than 1 h), and high
extraction efficiency. A large number of natural products, including poly-
phenols (Razende et al., 2017; Perez-Grijalva et al., 2018), have been
extracted by UAE.
Pressurized fluid extraction or accelerated solvent extraction or high-
pressure solvent extraction use an extraction system in which the samples
are mixed with suitable solvent(s) under conditions of high temperature
and pressure (Azmir et al., 2013; Xu et al., 2017). The main advantages
of PLE are reduced extraction time and reduced solvent amount, which
result in increased extraction efficiency.
In pulsed electric field (PEF), the samples are placed between two elec-
trodes, and generated pulse which increased the extraction of phytonutrients
from plant samples. The main advantage of using PEF is increased extraction
yield in a short extraction time. The process can also be used as a pre-
treatment of extraction materials.
Another sophisticated and advanced technique of phytonutrient extrac-
tion is enzyme assisted extraction (EAE), which increases the extraction effi-
ciency by enzymatic breakdown of plant material. This technique uses
enzymes (e.g., cellulase, α-amylase, pectinase, protease, etc.) to breakdown
the cell wall, and complex molecules, thus resulting in increased solvent per-
meability and extraction yield (Belwal et al., 2018; Azmir et al., 2013). The
use of an aqueous medium and application of a suitable temperature makes
EAE a suitable method for various industrial applications in the extraction of
phytonutrients.
Both conventional and nonconventional methods of extractions are still
used in the extraction of various phytonutrients. With the advancement of
technology in recent years, the nonconventional methods of extraction have
224 Tarun Belwal et al.

proven to have various advantages as compared with conventional methods.

However, many of these advanced techniques are also expensive, and a few
are still not scaled-up for large industrial applications. The choice of any par-
ticular extraction process also varies according to the chemical class of
phytonutrients of interest.

10.2.3 Purification methods

Once the extract is obtained in its desired form, the phytonutrients present
in the extract should be purified by the use of various purification methods.
There are many types of purification methods, such as crystallization, liquid-
liquid partition by the use of a separating funnel, and the application of chro-
matographic techniques.
Crystallization of major compounds from the extract or fraction is a
commonly used method before the development of advanced methods.
However, its efficiency depends on the physico-chemical nature of the
compound of interest, solvent, relative purity of the compound in mixture,
and so forth. Repeated crystallization (recrystallization) with a proper sol-
vent system may lead to purification of a compound in a few simple steps.
Liquid-liquid extraction, or partition, is another common economical
method to separate phytonutrients based on their solubility. Phytonutrients
are usually solubilized in a polar solvent (usually water), and then extracted
with nonpolar solvents (e.g., petroleum ether and n-hexane) by the use of a
separating funnel. Successive extraction by nonpolar solvents in the order of
their polarity results in the separation of compounds based on their polarity.
Most commonly used nonpolar solvents for extraction are n-hexane, ethyl
acetate, and n-butanol. However, the fractions obtained are usually mixtures
of compounds, and hence should be further purified by the use of chromato-
graphic methods.
Chromatography is a fundamental process in the separation of desired
phytonutrients from the extract (mixture of compounds), with the help
of a stationary phase and a mobile phase. Various chromatographic methods,
such as adsorption, partition, size exclusion, gel filtration chromatography,
and so forth, are used, depending on the physico-chemical nature of the
compound to be purified. Thin layer chromatography (TLC) is the classical
and most commonly used method to detect the presence of phytochemicals
in extracts, and their purity. TLC was widely used, even before the devel-
opment of more sophisticated instrumental chromatography methods, such
as high-performance liquid chromatography (HPLC). Using different
Optimization of extraction methodologies 225

derivatization agents, we can also obtain the qualitative information about

the nature of chemical compounds (Reich and Blatter, 2003). One limita-
tion of TLC is that it is difficult to isolate or purify compounds on the basis of
this method, as the sample applied is usually much smaller. However, for a
small amount of solvents, preparative-TLC can be used. In recent years,
development of a high performance-TLC (HPTLC) method has made it
possible to also perform quantitative analysis.
For the separation and purification of a large number of samples, column
chromatography is used, which utilizes a column-filled stationary phase. The
sample of interest to be separated is applied at the top of the stationary phase,
and continuously eluted with solvent, known as the mobile phase. Thus, the
mobile phase (solvent) passes through the stationary phase under the influ-
ence of gravity, or pressure, carrying the compounds at different speeds.
Adsorption chromatography can be divided into two types: a normal phase
and a reversed phase, based on the nature of the stationary phase. Use of nor-
mal phase adsorption chromatography is usually done for the separation of
nonpolar compounds, in which silica is commonly used as a stationary phase,
and nonpolar solvents (e.g., n-hexane, ethyl acetate, chloroform,
dichloromethane, etc.) are used as a mobile phase. Use of these solvents
in different ratios can result in the desired separation of compounds. In
reversed phase chromatography, octadecyl silica is used as a stationary phase,
and polar solvents (e.g., water, methanol or mixture of these solvents) are
used as a mobile phase.
In the past few decades, many advanced techniques of chromatography,
that is, HPLC, ultra-performance liquid chromatography (UPLC), and gas
chromatography (GC) have been developed in order to efficiently purify
natural products. These methods, coupled with mass spectrometry such as
LC-MS, HPLC-MS, UPLC-MS, and GC-MS, are widely used for rapid
purification/isolation and identification of these natural products.

10.3 Optimization strategies

Both extraction and purification methods have become highly
advanced in order to meet industrial goals. Among others, a majority of these
processes were developed to increase efficiency and to reduce process time,
as well as economic and environmental losses, which depend on the instru-
ment and on the process used. Optimization of the extraction and purifica-
tion processes is of great concern to meet desired product requirements.
Two strategies of optimization are being commonly used, which includes
226 Tarun Belwal et al.

the single-factor-at-a-time, and multiple-factor approaches. In the single-

factor-at-a-time approach, changes are made only in one factor at a time,
while keeping other factors constant. For example, extraction temperature
can be varied over its levels, while other factors, such as extraction time, sol-
vent concentration, pH, and so forth are kept constant. These experiments
are easy to perform, but require performing a large number of experiments,
and cannot distinguish between the different factors used in the studies. On
the other hand, a multi-factorial approach uses multi-factors at a time, with
changing levels. It is fast, and requires fewer experiments, and also deter-
mines the interactive effect between the factors. Some of the multifactor
optimization designs are discussed as follows.

10.3.1 Multi-factorial design

For optimization of extraction and purification conditions, a multi-factorial
design has been widely used. Multi-factorial design consists of two or more
independent variables that vary in their levels, and their effect was tested on a
single dependent variable. The independent variables (factors) include both
instrument and noninstrument factors. Instrumental factors vary with the
type of instrument used. For example, for MAE, microwave power and irra-
diation time would be the instrument factors, while in the case of UAE,
ultrasonic frequency, power/intensity could be the instrumental factors.
The noninstrumental factors include solvent concentration, volume, pH,
particle size, and so forth, which mostly depend on the plant material and
the compound of interest. In multi-factorial experiments, different factors
are involved with different numbers of levels. For example, when a design
is denoted as 23 factorial, this identifies the number of factors (3); how many
levels each factor has (2); and how many experimental conditions there are
in the design (23 ¼ 8). Similarly, a 25 design has five factors, each with two
levels, and 25 ¼ 32 experimental conditions; and a 32 design has two factors,
each with three levels, and 32 ¼ 9 experimental conditions.
The optimization experiments were designed for the determination of
extraction and purification conditions in order to understand recovery of
phytochemical content at its best level. Two types of multi-factorial designs
were used (i) Plackett-Burman design (PBD) for the screening of significant
factors, followed by (ii) central composite design (CCD) or Box-Behnken
design (BBD) for optimization and validation of experimental conditions
(Fig. 10.3). Other optimization models include an artificial neural network,
three-level factorials, and hybrid, pentagonal, and hexagonal designs.
Optimization of extraction methodologies 227

‘N’ number of factors

Significant factors PBD design

RSM models

Opmizaon of factors BBD/CCD design,

Taguchi design

Validaon of the model

Fig. 10.3 Systematic representation of multi-factorial optimization design.

A number of extraction and purification methods have been optimized

based on these models (Table 10.2). Both CCD and BBD involve response
surface methodology (RSM), which generates 3D response surface graphs
based on the response value obtained during the experimental runs. One
effective mathematical and statistical tool is RSM, in which use of second
order polynomial design for multi-factor studies is applied (Myers et al.,
2016). However, Taguchi designs are saturated orthogonal arrays with all
main effects, and no interactions. Using a linear effect, one can estimate main
effects, and also estimate certain interactions. The Taguchi design has the
advantage of using smaller fractions of a full factorial design, such as a
half-fraction, quarter-fraction, or eighth-fraction, and thus reduces the cost
and time. These designs not only use fewer experimental runs, they are also
helpful in determining an interactive effect between the factors. Moreover,
these designs give total control to each and every factor to obtain the desired
output.

10.3.1.1 Plackett-Burman design

Plackett-Burman design (PBD) is a multi-factorial experimental design
which was presented by Robin L. Plackett and J.P. Burman in 1946.
PBD identify the most significant factors early in the experimentation phase.
In PBD, basically the investigation of the dependency of measured quantity
is done on a number of independent variables, each taking “L” levels in such
a way that the variance of the estimates of these dependencies are minimized
using a limited number of experiments. Interactions between the factors
were considered negligible. The solution to this problem is to find an
228 Tarun Belwal et al.

Table 10.2 List of some of the extraction and purification optimization experiments
Optimization
Plant and its part Phytonutrients method References
Acai berry, Phenolics and 23 factorial design de Moura et al. (2018)
Blueberry, Goji antioxidants
berry
Wheat whole grain Antioxidant CCD Liyana-Pathirana and
and bran activity Shahidi (2005)
Grape seeds Phenolics and CCD Ghafoor et al. (2009)
anthocyanins
Berberis asiatica fruits Phenolics and CCD Belwal et al. (2016)
antioxidants
Berberis jaeschkeana Phenolics and PBD and CCD Belwal et al. (2017)
fruits antioxidants
Berberis asiatica leaves Phenolics and PBD and BBD Belwal et al. (2017)
antioxidants
Paris polyphylla Phenolics and PBD and CCD Mohd et al. (2018)
leaves antioxidants
Rheum Phenolics and BBD Pandey et al. (2018)
moorcroftianum antioxidants
rhizome
Acer truncatum leaves Phenolics and CCD Yang et al. (2017)
anthocyanins
Apple pomace Phenolics BBD Bai et al. (2010)
Crataegus laevigata Chlorogenic BBD Cui et al. (2011)
acid
Zea mays Anthocyanins BBD Yang and Zhai (2010)
Tomato and Vitamin E BBD Lee et al. (2000)
Broccoli
Curcuma longa Curcumin Taguchi L9 Mandal et al. (2008)
rhizome orthogonal
design
Candlenut Oil Taguchi L9 Subroto et al. (2017)
orthogonal
design
Red cabbage Anthocyanins Taguchi L9 Ravanfar et al. (2015)
orthogonal
design
Pomegranate rinds Natural dye CCD and ANN Sinha et al. (2012)
Coptis chinensis Alkaloids CCD Teng and Choi (2014)
rhizome
CCD, central composite design; BBD, Box-Behnken design; PBD, Plackett-Burman design; ANN, arti-
ficial neural network.
Optimization of extraction methodologies 229

experimental design in which each combination of levels for any pair of fac-
tors appears the same number of times, throughout all the experimental runs.
For example, in MAE optimization experimentation, PBD was applied
to different screening factors, i.e., microwave power, irradiation time, sam-
ple to solvent ratio, and solvent pH and concentration. These factors were
found to significantly (P < 0.05) affect phytochemical extraction from the
plant source. In this experiment, lower (1) and higher (+1) levels of each
factor were tested, and a total of 12 experimental runs were performed. The
PBD experiment followed a first-order polynomial equation (10.1):
X
Y ¼ β0 + β i Xi (10.1)
i¼1

where, Y is the response variable, β0 is intercept, βi is linear regression coef-

ficient for ith factor, and Xi is the level of the independent factor. In this
Plackett-Burman experimental design, no interaction and quadratic effects
were considered. Based on their F-value, factors that significantly (P < 0.05)
affect the response value were selected, and then screened further at different
levels, as well as optimized for linear, quadratic, and interactive effect using
second-order polynomial design.

10.3.1.2 Central composite design

A Box-Wilson central composite design, also known as central composite
design (CCD), works on embedded factorial or fractional factorial design
with center points that are augmented with a group of star points which
allow curvature estimation (Fig. 10.4A). It is usually applied to study the
effect of dependent factors at higher or lower levels in a single run. The fac-
tor varies with a maximum of five and a minimum of three levels, with an α

Fig. 10.4 Factors allocation in (A) Central Composite design and (B) Box-Behnken
design.
230 Tarun Belwal et al.

value (star points) that depends on required desired properties for the design,
as well as the number of factors involved. For face centered CCD, α ¼ 1 as
the star points (α) are at the center of each face of the factorial space. This
design needs three levels of each factor. While for rotatable CCD, the star
points (α) are some distance from the center, depending on the desired prop-
erties of the design, as well as the number of factors in the design. The star
points establish new extremes for the low and high settings for all factors, and
require five levels for each factor.
The CCD consist of different sets of experiments that depend on the
number of factors considered for the determination of phytochemicals. Each
factor is coded at three (1, 0, +1) or five levels (α, 1, 0, +1, +α),
depending on the experimental conditions and response interest. To fit
the experimental data in a second order polynomial equation, regression
analysis was conducted with the following Eq. (10.2):

X
k X
k X
k X
k1
Y ¼ β0 + β i Xi + βii Xi2 + βij Xi Xj (10.2)
i¼1 i¼1 i¼1 j¼i + 1

where, Y is the response variable, Xi and Xj are the independent variables,

and k is the number of tested factors. The regression coefficient is defined as
β0 for intercept, βi for linear, βii for quadratic, and βij for the cross-product
term. 3D graphs were generated using regression coefficients, and analysis of
variance was conducted to find the significant effect of the model terms (lin-
ear, quadratic, and cross product). Model F-value, lack of fitness, and the
regression coefficient were also estimated for each response model.

10.3.1.3 Box-Behnken design

The Box-Behnken design (BBD) is an independent quadratic design in
which the factor combinations are at the midpoints of edges of the process
space, and at the center (Fig. 10.4B). The requirement of BBD is that here,
three levels of each factor are needed, which results in fewer runs as com-
pared with CCD. However, because BBD does not involved experimental
runs where all the factors are at their higher or lower levels, it may be useful
to avoid experiments under extreme conditions, for which unsatisfactory
results might occur.
The factors considered for BBD include the same as those for CCD, and
each factor is coded at its three level (i.e., 1, 0, and 1) which represent
lower, middle, and higher values, respectively. In order to fit experimental
Optimization of extraction methodologies 231

data in a second order polynomial equation, regression analysis was

conducted, similar to CCD (Eq. 7.2).
Various tools have been used for optimization experiments; these
includes Design Expert, Sigma XL, Minitab, and Comprehensive R.

10.4 Trends in phytonutrient extraction and

purification
With the increased demand for functional foods, or neutraceuticals, in
the market, there is an ever-increasing interest in the extraction and purifi-
cation of phytonutrients. Many of these advanced extraction techniques are
used for the extraction and isolation of natural products to be used as
neutraceuticals.
To get a clear idea about the recent research trend on extraction and
purification related to phytonutrients, we performed a literature survey
on Scopus (www.scopus.com). The search parameters are given in
Table 10.3. Table 10.4 summarizes the number of research papers dealing
with different compound classes and plant parts. Among the search items
given in Table 10.4, flavonoids were the most studied class of compounds

Table 10.3 Parameters used during the search for articles in Scopus (www.scopus.com)
Search parameters Operational settings
Search term text (i) “Anthocyanins” AND “Roots,” “Anthocyanins”
AND “Rhizomes,” and so on for individual
parameters (Table 10.4 for chemical class and plant
part)
(ii) “Microwave assisted extraction” AND “Antho-
cyanins” and so on for individual parameters
(Table 10.5 for Patents)
(iii) “Microwave assisted extraction” AND “optimi-
zation” and so on for individual parameters
(Table 10.6 for optimization)
Search field type Article title, abstract, keywords
Date range All years to present
Subject Areas All
Document type Patents for (i) and articles for others
Operator used between AND
two search items
232 Tarun Belwal et al.

Table 10.4 No of papers dealing with different phytonutrients compound classes and
plant parts
Chemical classes Roots Rhizomes Leaves Bark Fruits Flowers
Anthocyanins 1157 51 3256 163 7743 2365
Carotenoids 2097 59 5306 93 6538 997
Flavonoids 6052 865 15,957 2735 11,224 3957
Phenols 4903 629 10,849 1890 9833 1730
Lignans 604 137 703 440 719 94
Terpenoids 1384 229 3210 634 964 700
Alkaloids 6666 800 8952 3046 2288 1253
Source: Scopus (retrieve on February 19, 2018).

that were mainly isolated from the fruits, leaves, and roots. Phenolic com-
pounds were also reported to be mostly isolated from leaves, followed by
fruits. Another class of phytonutrients, anthocyanins, was mostly isolated
from fruits, followed by leaves. These results give some idea about the abun-
dance of these phytochemicals in different plant parts.
Table 10.5 summarizes the number of research articles dealing with dif-
ferent compound classes and advanced extraction techniques. MAE and SFE
were found to be the most widely used methods for the extraction of phy-
tochemicals belonging to classes of flavonoids and phenols. The direct com-
parison may be difficult, as some of these techniques have been developed
recently, and some have wide industrial applications. However, it may be
interesting to follow this trend in the research on phytonutrients in the
future to see the developments in this field.

Table 10.5 No of papers dealing with different phytonutrients compound classes and
nonconventional extraction techniques
Chemical classes MAE SFE UAE PLE PEF EAE
Anthocyanins 60 54 24 26 3 11
Carotenoids 28 223 9 34 2 16
Flavonoids 297 265 130 72 0 22
Phenols 314 475 83 118 1 20
Lignans 11 27 7 7 0 1
Curcumin 15 20 4 3 0 2
Terpenoids 20 55 6 3 0 1
Alkaloids 68 90 18 14 1 2
MAE, microwave assisted extraction; UAE, ultrasonic assisted extraction; SFE, supercritical fluid extrac-
tion; PEF, pulsed electric field; PLE, pressurized liquid extraction; EAE, enzyme assisted extraction.
Source: Scopus (retrieve on February 19, 2018).
Optimization of extraction methodologies 233

Table 10.6 No. of papers published on optimization of different extraction techniques

Extraction method Short form No. of papers
Microwave assisted extraction MAE 663
Supercritical fluid extraction SFE 915
Ultrasonic assisted extraction UAE 309
Pressurized liquid extraction PLE 203
Pulsed electric field extraction PEF 2
Enzyme assisted extraction EAE 54
Source: Scopus (retrieve on April 29, 2017).

Table 10.6 summarizes the number of papers published on the optimi-

zation of different extraction techniques. Most of the optimization studies
were focused on SFE, followed by MAE. A large number of papers publi-
shed on the use and optimization of SFE and MAE suggest that these two
techniques have several advantages over other techniques, and have wider
application possibilities.

10.5 Conclusion and future prospects

This chapter emphasizes the extraction and purification techniques for
phytonutrients and their optimization strategies. Optimization of these pro-
cess steps in phytonutrient research is an important task in the process of
ensuring product efficiency, as well as meeting economic and environmental
goals. Various optimization models have been tested on extraction and puri-
fication methods for phytonutrients. However, more research is needed on
the optimization of biological activity and physico-chemical properties of
phytonutrients. Apart from this, promising research areas can be developed
based on combined optimization models for extraction and purification
techniques, which can lead to better results. The emphasis should also be
placed on validation and repeatability of the optimization model. It has also
been seen that most of the works on optimization of extraction and purifi-
cation techniques for phytonutrients are presently at the lab level or pilot
scale, which needs to be upscaled to the industrial level for better under-
standing of the process factors during scale-up process. Moreover, efficient
extraction and purification methods that can meet the requirements of green
technologies must be addressed in the future.
234 Tarun Belwal et al.

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Further reading
Cheah, E.L., Heng, P.W., Chan, L.W., 2010. Optimization of supercritical fluid extraction
and pressurized liquid extraction of active principles from Magnolia officinalis using the
Taguchi design. Sep. Purif. Technol. 71 (3), 293–301.