Separation and Purification Technology 157 (2016) 169–178

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Microwave assisted extraction of flavonoids from Terminalia bellerica:

Study of kinetics and thermodynamics
R. Yedhu Krishnan, K.S. Rajan ⇑
Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology, SASTRA University, Thanjavur 613401, India

a r t i c l e i n f o a b s t r a c t

Article history: Microwave-assisted solid–liquid extraction (MAE) of flavonoids from Terminalia bellerica Roxb. has been
Received 21 September 2015 investigated to understand the influence of solvent-to-feed ratio (5–40 mL/g) and temperature
Received in revised form 7 November 2015 (40–100 °C) on kinetics and thermodynamics of extraction. The flavonoid concentration–time data were
Accepted 25 November 2015
analysed using a second order kinetic model to determine extraction constant. A diffusion model was
Available online 2 December 2015
utilized to determine diffusion coefficient taking into account of both washing and diffusion phases
together. Solvent-to-feed ratio and temperature were observed to show a significant effect on flavonoid
Keywords:
yield, extraction rate and effective diffusion coefficient with the highest solvent-to-feed ratio and tem-
Microwave assisted extraction
Terminalia bellerica
perature providing the maximum flavonoid yield of 25.21 mgQE/g using water as the solvent. An empir-
Flavonoids ical equation has been developed to predict extraction kinetics. Thermodynamic parameters revealed
Kinetics MAE process to be spontaneous and endothermic. The kinetic and diffusion models provided valuable
Effective diffusion coefficient insights into the microwave assisted extraction process. T. bellerica was observed to be a good source
Thermodynamics of flavonoids. Therefore, MAE is a suitable method for extraction of flavonoids from T. bellerica when
water alone is to be used as the solvent, as in the case of traditional Indian medicinal preparations.
Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Flavonoids are polyphenolic plant pigments that are ubiquitous

Solid–liquid extraction involves the recovery of solute from the
solid media using a liquid solvent [1,2]. The use of microwave radi-
ation for mass transfer intensification is well-known [2]. In micro-
wave assisted solid–liquid extraction, the extraction is accelerated
due to the changes in cell anatomy caused by the impact of micro-
wave radiation. The accelerated extraction may be attributed to
synergistic action of temperature and concentration gradients
operating in the same direction [3,4]. It is pertinent to note that
in microwave assisted solid–liquid extraction, the heat is dissi-
pated directly within the moisture-laden solid matrix causing the
sudden disruption of the same [5]. However, in the conventional
solid–liquid extraction methods, heat transfer takes place from
external cell surfaces to the internal core [3].
Microwave assisted solid–liquid extraction (MAE) is a widely
used technique for the extraction of valuable bioactive components
from plant materials [6–10]. MAE is superior compared to other
conventional methods of extraction like Heat Reflux Extraction
(HRE) or Soxhlet extraction with respect to extraction time, solvent
consumption, the final yield of purified product, etc. [5,11–13].
⇑ Corresponding author.
E-mail address: ksrajan@chem.sastra.edu (K.S. Rajan).
to plant cells. These are antioxidants and free radical scavengers
protecting against the oxidative reactions taking place in the body.
The average daily intake of these secondary metabolites from nat-
ural sources for humans is 1–2 g per day [14]. Research on flavo-
noids has gained significant importance as they act as functional
compounds with the potential to serve as derivatives of new drugs.
Also, beneficial effects of flavonoid-rich natural products have been
established [14]. Supplementing processed foods with flavonoids is
being practiced to warrant sufficient daily intake to enhance the
immune system [15]. One such important plant widely used in
the treatment of various ailments and a rich source of flavonoids
and other bioactive compounds is Terminalia bellerica Roxb.
T. bellerica Roxb. (T. bellerica) is a deciduous medicinal tree that
is seen mostly in the moist valleys, lower hills of India and in other
parts of South East and Central Asia. It belongs to the Combretaceae
family and is a rich source of bioactive constituents [16]. Various
parts of the tree are used for different therapeutic purposes. How-
ever, the dry fruits of T. bellerica possess maximum health benefits
[17]. Traditionally it is one of the principal constituents of the
Ayurvedic formulation Triphala, where it is used as an expectorant
[18]. Ethanolic extract of T. bellerica was determined to be effective
against several pathogenic organisms like P. vulgaris, E. coli,
B. subtilis and S. aureus [19]. Separation and purification of vital

http://dx.doi.org/10.1016/j.seppur.2015.11.035
1383-5866/Ó 2015 Elsevier B.V. All rights reserved.
170 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178
bioactive constituents from the dried fruits of T. bellerica will open
new inroads in the pharmaceutical, chemical, food and processing
industries.
For the development of an efficient MAE, a study on the kinetics
and thermodynamics parameters are pivotal [20–22]. Several mod-
els have been proposed to understand the influence of operating
parameters on the extraction kinetics [23]. These include models
based on first order and second order behaviour [24,25], modified
Fick’s law [26] and other empirical models [27–29]. The study on
thermodynamic parameters of extraction helps in determining
the spontaneity of extraction process [30].
The water extract of T. bellerica is utilized in the preparation of
several Ayurvedic medicines such as Lauha bhasma [18]. Hence the
intensification of extraction of flavonoids from T. bellerica using
MAE with water alone as the solvent has immediate industrial
applications. Therefore, the present work has been carried out to
study the kinetics, effective diffusion coefficient and thermody-
namics of extraction of flavonoids from T. bellerica using MAE with
water as the solvent. The experiments were designed to accommo-
date a wide variety of operating conditions. A specific extraction
model for the kinetics of MAE of flavonoids from T. bellerica is
not reported in the literature to the best of our knowledge. The
effective diffusion coefficient for MAE of flavonoids from T. bellerica
has also been determined. The study assumes crucial significance
as thermodynamic properties and spontaneity of MAE of flavo-
noids from T. bellerica has not been reported previously in the
literature.
2. Materials and methods
2.1. Materials and reagents
Dried fruits of T. bellerica were obtained from the market and
authenticated by CARISM, SASTRA University, Thanjavur, India.
These fruits were ground in a domestic mixer grinder. The powder
was screened in a rotary sieve shaker to select particle sizes
between BSS
200/+300 mesh size (average particle size –
0.064 mm). Aluminium nitrate and potassium acetate used for
the Total Flavonoid Content (TFC) assay were purchased from Hi
Media Ltd., Mumbai, India. Quercetin was purchased from Aldrich
Fig. 1. Schematic diagram of experimental setup for microwave assisted extraction.
Chemical Company, USA. DPPH was procured from SRL Chemicals,
India. Freshly prepared distilled water was used as the solvent. The
chemicals were used as obtained with no further purification.
2.2. Microwave assisted extraction
A modified microwave oven (R-219T (S)/(W), SHARP, Japan) of
22 l capacity was used for microwave assisted extraction. The
microwave oven was equipped with a timer to control the duration
of on–off cycles of microwave irradiation. A ‘J’ type thermocouple
(Microsensors, India) was used as the temperature sensor. A tem-
perature indicator cum controller with a resolution of ±0.1 °C
(Microsensors, India) was utilized to monitor the temperature dur-
ing extraction, feedback from which was used to control the dura-
tion of on–off cycles. A hole was drilled in the outer casing to
mount a reflux condenser onto the oven. Temperature was mea-
sured and controlled using a digital temperature indicator cum
controller. A magnetic stirrer was attached to the bottom of the
microwave oven for ideal mixing. For the purpose of MAE of flavo-
noids from T. bellerica dried fruits, a double-necked glass vessel
was filled with a measured quantity of powdered fruit samples
and the solvent (water). This mixture was placed in the centre of
the microwave oven and was subjected to different durations of
exposure. Samples were withdrawn at specified time intervals. A
schematic diagram of experimental setup for MAE is shown in
Fig. 1.
2.3. Conventional solid–liquid extraction (CE)
The extraction of flavonoids from T. bellerica fruit powder was
performed by conventional heat-reflux extraction. The tempera-
ture in the reaction vessel was monitored using a digital tempera-
ture indicator. The measured quantity of dried fruit sample was
mixed along with the required volume of solvent according to
the experimental design.
An exhaustive extraction of T. bellerica was performed by CE
using different solvents for the determination of Total Flavonoid
Content in the material, which is independent of the type of
extraction. CE was performed with nonpolar (chloroform) and
polar solvents (methanol, water) in series, to ensure that the entire
 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178
flavonoid content present in the fruit was extracted for the estima-
tion of its quantity [31]. Accordingly, 12 h extraction was per-
formed initially with chloroform as the solvent. This was
followed by successive extractions with methanol and water
respectively, for 12 h each.
2.4. Experimental design
A full factorial design was used to study the effects of solvent-
to-feed ratio (S) and temperature (T) for the extraction of flavo-
noids from T. bellerica fruits. Four levels were used for each of
the two factors: solvent-to-feed ratio (5, 10, 20, 40 mL/g) and tem-
perature (40, 60, 80, 100 °C). The experiments were carried out in
triplicates. Accordingly 48 experiments (24  3) were carried out as
per the full factorial design. The table of experimental runs is not
provided here to maintain brevity. Temperature above 100 °C
(boiling point of solvent) was not considered for the study. The
range of solvent-to-feed ratio was fixed between 5 and 40 mL/g
as this range was optimum for the preparation of extracts used
in the preparation of Lauha bhasma [18].
2.5. Analytical method
2.5.1. Total Flavonoid Content
The Total Flavonoid Content was estimated using aluminium
nitrate assay [32,33]. 500 lL of the diluted sample was mixed thor-
oughly with 0.1 mL of 10% aluminium nitrate, 0.1 mL of 1 M potas-
sium acetate and 4.3 mL of distilled water and allowed to stand at
room temperature for 40 min. A change in colour of the prepared
samples from colourless to yellow indicated the presence of flavo-
noids. Absorbance was measured at 415 nm (Lambda 25, Perkin
Elmer, USA) against a reagent blank prepared similarly without
the extract. A standard curve was prepared using quercetin and
the results were expressed as mg quercetin equivalent per mL of
solvent (mgQE/mL). All the assays were repeated three times and
the average values were used for further analysis.
2.5.2. Test for antioxidant activity
The antioxidant activity of aqueous extracts of T. bellerica was
studied using 2,2-diphenyl-1-picryl-hydrazil radical (DPPH) [34].
The extracts were vacuum dried in a rotary evaporator (R-210,
Buchi) and further air dried in a hot air oven at 100 °C for 1 h.
The dried extracts were weighed and reconstituted to known con-
centrations. 0.1 mL of the extract and 3.9 mL methanolic solution
of DPPH (5 mg in 100 mL methanol) were treated together and
incubated at room temperature for 30 min in dark and the absor-
bance was measured at 515 nm (Lamda 25, Perkin Elmer, USA)
against distilled water. Similarly, a control solution was prepared
with 0.1 mL distilled water and 3.9 mL methanolic solution of
DPPH. The radical scavenging activity of the extracts is expressed
as percentage inhibition of DPPH as follows:
DPPH radical scavenging activity ð%Þ
 
Absorbance of sample at 515 nm
¼ 1
 100
Absorbance of control at 515 nm
2.6. Gas chromatography – mass spectrometry (GC–MS)
The representative extract obtained using MAE and CE were
dried and reconstituted in ethanol for GC–MS analysis. GC–MS
analysis was carried out using a gas chromatograph (PerkinElmer
Clarus 500) with a 250 lm diameter and 30 m long Elite-5 capillary
column (5% phenyl/95% dimethylpolysiloxane) equipped with a
mass selective detector in the electron ionization mode. About
2 lL of the sample was injected for the analysis. Helium was used
171
as the carrier gas and supplied at the flow rate of 1 mL/min, with a
split ratio of 1:10. The oven temperature was increased from 60 °C
to 150 °C at the ramp rate of 6 °C/min and held at 150 °C for 2 min.
This was followed by temperature increase to 280 °C at the rate of
4 °C/min and held at the same temperature (280 °C) for 5 min. The
injector was maintained at 280 °C. The mass spectra were recorded
at 70 eV over a mass-to-charge ratio range of 40–450 amu. The
chemical compositions were identified by direct comparison of
their mass spectral pattern in NIST 2005 mass spectral library.
2.7. Scanning electron microscopy
The surface morphology of the feed material separated after
MAE and CE were investigated using a field emission scanning
electron microscope (JSM 6701F, JEOL, Japan). The samples were
sprinkled on the carbon pasted stub, following which a thin layer
of gold was sputter coated to render the surface conductive.
2.8. Mathematical tools
2.8.1. Second-order kinetic model
A general second-order model has been used to study the kinet-
ics of solid–liquid extraction. The second order model provides an
acceptable representation of solid–liquid extraction, as evident
from its prominent use in modelling extraction [25,35,36]. The
parameters in the second order kinetic model can easily be eluci-
dated from the extraction kinetics, using the analytical solution
obtained by integral analysis. The same model has been extended
to study the MAE of flavonoids from T. bellerica also. The second-
order kinetic model can be represented as:
dC t
¼ kðC s
C t Þ2 ð1Þ
dt
where Ct is the concentration of flavonoids (mgQE/mL) at any time t,
Cs is the saturation concentration of flavonoids (mgQE/mL) and k is
the second order rate constant (mL/mgQE min).
Solving Eq. (1) with the boundary conditions as Ct|t=0 = 0 and
Ct|t=t = Ct gives
C 2s kt
Ct ¼ ð2Þ
1 þ C s kt
Eq. (2) can be rewritten as Eq. (3) and subsequently reduced to
Eq. (4) as follows, where ‘h’ is the product of ‘k’ and C 2s .
t 1 t
¼ þ ð3Þ
C t kC 2s C s
t 1 t
¼ þ ð4Þ
Ct h Cs
‘h’ is the initial extraction rate (mgQE/mL min). A plot of t/Ct vs t can
be drawn to determine Cs, k and h.
The effect of temperature on extraction rate constant provides
information on the activation energy that must be overcome for
extraction. The extraction rate constant is related to the activation
energy (Ea) and temperature (T) as follows:

Ea
k ¼ k0  e RT ð5Þ
A plot of ln(k) vs 1/T, yields a straight line with (
Ea/R) as the
slope, from which activation energy can be determined.
2.8.2. Determination of effective diffusion coefficient and Biot number
To study the effective diffusion coefficient of flavonoids from
T. bellerica, the diffusion model based on Fick’s second law was
used. Notable assumptions made while applying the model for
studying extraction process are listed below:
172 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178

(a) The T. bellerica dried fruit particles were considered to be 3. Results and discussion
spherical in geometry with an average diameter of 0.064 mm.
(b) The liquid was well mixed with the help of magnetic stirrer 3.1. Kinetics of MAE
attached to the microwave oven and hence resistance in the
liquid phase was negligible. The appropriate conditions for rapid extraction of flavonoids
(c) Degradation of flavonoids extracted by microwave from T. bellerica by MAE can be identified through the study of
irradiation was absent. extraction kinetics. Fig. 2 shows that the variation of flavonoid con-
centration in the liquid with progress in extraction time at a
Fick’s second law for extraction of flavonoids from spherical solvent-to-feed ratio of 40 mL/g. It is evident from Fig. 2 that flavo-
particles is as follows [37]: noid concentration increased rapidly with increasing time during
   the initial phase of MAE, followed by more-controlled increase
@M 1 @ @M
¼ De 2 r2 ð6Þ and near-saturation during later stages of extraction. This might
@t r @r @t
be attributed to rapid dissolution of solute on particle surface
where M is the flavonoid concentration in T. bellerica fruit particle through washing and followed by solute removal by diffusion [41].
(kg/m3), r is the distance from the centre of spherical T. bellerica The second order model (Eq. (2)) was found to describe the
fruit particle (m), De is the effective diffusion coefficient of kinetics of MAE of flavonoids from T. bellerica satisfactorily for all
flavonoids and t is time (s). experimental conditions as shown in Fig. 3 with R2 of 0.999. The
The solution of Eq. (6) using the initial and boundary conditions average absolute relative deviation (AARD) and the relative stan-
is [38]: dard deviation between the experimental data and those predicted
  by second-order kinetics are 0.38% and 0.62% respectively.
Yt 6 D e p2 t
¼ 1
2 exp
ð7Þ
Ys p R 2
3.2. Effect of solvent-to-feed ratio and temperature
where Yt is the total flavonoid (mgQE/g) transferred from T. bellerica
fruit at time t, Ys is the total flavonoid transferred (mgQE/g) at sat- Fig. 4a shows the effect of solvent-to-feed ratio (S) on the equi-
uration. Accordingly the total flavonoid yields at time t, and at sat- librium yield when MAE was carried out at 40 and 100 °C. It is
uration are Yt and Ys respectively. The effective diffusion coefficient evident from Fig. 4a that the equilibrium yield increased with
can be calculated from time – yield data using Eq. (7). solvent-to-feed ratio (S) at both the temperatures. The equilibrium
The relative magnitudes of external and internal resistances to yield was found to increase rapidly at lower solvent-to-feed ratios
mass transfer can be ascertained by determining Biot number (S < 20), as compared to that at higher solvent-to-feed ratios
(Bi). The Biot number for solid–liquid extraction is given by [39]: (S > 20) and exhibited a power-law dependency on solvent-to-
Dp K T feed ratio with an exponent 6 1 (0.2431 at 100 °C). The moisture
Bi ¼ ð8Þ present in the cell matrix results in the sudden rise in temperature
De
locally due to the absorption of microwave energy [3] causing cell
where KT is the mass transfer coefficient (m/s) and Dp is the size of rupture. Microwave absorption also results in the increase of solu-
particle (m). bility [42]. Hence increased solubility and improved solute–solvent
The mass transfer coefficient (KT) can be determined from contact due to the rupture of the cell enhances the mass transfer
extraction kinetics using the following equation [40]: from solid to liquid phase. The extraction rate constant (k) too
Cs KtA increased with increase in solvent-to-feed ratio (Fig. 4b).
ln ¼ t ð9Þ The effect of solvent-to-feed ratio (S) on saturation concentra-
Cs
Ct VL
tion of flavonoids (Cs) and initial extraction rate (h) and from
However, it should be noted that Eq. (15) holds good only till T. bellerica is shown in Fig. 5. The saturation concentration
t 6 1/kCs [39]. (Fig. 5a) was found to be highest at the lowest solvent-to-feed ratio
and then decreased progressively with the increase in solvent-to-
2.8.3. Thermodynamics of MAE feed ratio, as generally observed in solid–liquid extraction [43].
Thermodynamic analysis of extraction of solute from solid Despite higher equilibrium yields at higher solvent-to-feed ratios,
using extraction has been carried out through determination of the saturation flavonoid concentration was lower. This could be
DG°, DH° and DS°.
Van’t Hoff equation [41] can be used to determine DH° and DS°
as follows:
DH DS
ln K e ¼
þ ð10Þ
RT R
where ‘Ke’ is the equilibrium constant for leaching and is defined as
the ratio of amount of solute extracted to the amount of solute
remaining unextracted [41]. In the present case, it is the ratio of
the amount of flavonoids extracted to the amount of flavonoids
remaining unextracted as given below:
Ys
Ke ¼ ð11Þ
Y max
Y s
where Ys is the total flavonoid yield (mgQE/g) extracted using water
as the solvent at a temperature T (K) and Ymax is the total flavonoid
yield (mgQE/g) determined after an exhaustive extraction using
chloroform, methanol and water as the solvents.
DG° is calculated from DH° and DS° as follows [41]:
Fig. 2. Effect of extraction time on the concentration of flavonoids during MAE
DG ¼ DH
T DS ð12Þ carried out at solvent-to-feed ratio of 40 mL/g.
 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178 173

Fig. 3. Suitability of second order model for the kinetics of MAE of flavonoids from T. bellerica.

higher at lower solvent-to-feed ratios (Fig. 5b) due to higher flavo-

noid saturation concentration. It can be noted from Eq. (4) that ini-
tial rate of extraction is directly proportional to the extraction rate
constant and square of saturation concentration, indicating that
saturation concentration has a higher influence on the initial rate
of extraction. Hence the reduction in saturation concentration at
higher solvent-to-feed ratios resulted in reduction in initial extrac-
tion rate also. The effect of reduction in saturation concentration
on initial extraction rate at higher solvent-to-feed ratios was partly
offset by increase in extraction rate constant with solvent-to-feed
ratio.
The effect of temperature (T) on saturation concentration is
shown in Fig. 6. With increase in temperature, saturation concen-
tration was found to increase at all solvent-to-feed ratios investi-
gated. The increase in saturation concentration at higher
temperatures may be attributed to weaker solute–solute and
solute–solid interactions. The influence of temperature on satura-
tion concentration was more predominant at lower solvent-to-
feed ratio. The extraction rate constant (Fig. 7a) too increased with
increase in temperature, possibly due to higher thermal energy for
solute diffusion. The initial extraction rate (h) is the product of
extraction rate constant (k) and the square of saturation concentra-
tion (Cs), as evident from Eqs. (3) and (4). With the increase in tem-
perature, both the saturation concentration (Fig. 6) and extraction
rate constant (Fig. 7a) were increased. Hence, the initial extraction
rate also increased with temperature (Fig. 7b).
Regression analysis was carried out to develop empirical corre-
lations for prediction of ‘h’ and ‘Cs’ and hence the extraction kinet-
ics, as a function of temperature and solvent-to-feed ratio. The
physical phenomena occurring during microwave irradiation is
challenging to model and hence the development of empirical cor-
relations becomes essential. The regression analysis of T–S–h and
T–S–Cs data carried out using Minitab 14 yielded the following
equations:
Fig. 4. Effect of solvent-to-feed ratio on (a) equilibrium yield and (b) extraction rate
constant.
1:05
40 6 T 6 100  C&5 6 S 6 40 mL=g
1:12
h ¼ 1:0757ðTÞ ðSÞ ð13Þ
0:232
0:777 
C s ¼ 3:6327ðTÞ ðSÞ 40 6 T 6 100 C&5 6 S 6 40 mL=g ð14Þ
attributed to the higher volume of solvent used at higher solvent-
to-feed ratios. The initial extraction rate was also observed to be Substituting Eqs. (13) and (14) in Eq. (4), leads to
174 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178

Fig. 5. Effect of solvent-to-feed ratio on (a) saturation concentration and (b) initial Fig. 7. Effect of temperature on (a) extraction rate constant and (b) initial
extraction rate. extraction rate.

0.996. Eq. (15) in conjunction with Eq. (14) and Eq. (1) can also
be utilized to determine the extraction time required to achieve
a desired concentration of flavonoids in extract using MAE.
The extraction rate constant can be used to determine the acti-
vation energy of extraction (Eq. (5)). Activation energy indicates
the energy barrier that has to be overcome to achieve solute trans-
fer. Solute–solute cohesive and solute–solid adhesive interactions
are the major contributors to activation energy for extraction
[44]. At higher temperatures, the solute–solute and solute–solid
interactions become quite weak, which results in increased extrac-
tion rate constant at higher temperatures for MAE [44]. Fig. 8
shows the plot of ln k versus the reciprocal of absolute tempera-
ture. The activation energy for MAE of flavonoids from T. bellerica
was found to be 12.07 kJ/mol in the temperature range of
40–100 °C at the solvent-to-feed ratio of 40 mL/g.

3.3. Effective diffusion coefficient and mass transfer coefficient

Fig. 6. Effect of temperature on saturation concentration. The effective diffusion coefficient and mass transfer coefficient
for extraction of flavonoids from T. bellerica using MAE were esti-
t
Ct ¼ 40 6 T 6 100  C&5 mated using Eqs. (7) and (9). The diffusion model (Eq. (7)) repre-
1
1:0757ðTÞ1:12 ðSÞ
1:05
þ t
3:6327ðTÞ0:232 ðSÞ
0:777 sents the MAE of flavonoids from T. bellerica as evident from the
6 S 6 40 mL=g ð15Þ closeness of prediction flavonoid concentration – time data with
those of experiments (Fig. 9).
Eq. (15) predicts Ct–t data for the temperature range of Table 1 summarizes the variation of effective diffusion coeffi-
40–100 °C, solvent-to-feed ratio range of 5–40 mL/g and time cient, mass transfer coefficient and Biot number with the temper-
range of 2–120 min with average absolute relative deviation ature at solvent-to-feed ratio of 40 mL/g. It can be observed from
(AARD) of 3.4%, relative standard deviation of 3.50% and R2 of Table 1 that effective diffusion coefficient and mass transfer
 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178
Fig. 8. Determination of activation energy for extraction.
coefficient increased with increasing temperature. While the
increase in diffusion coefficient may be attributed to increased
thermal energy at higher temperatures, the increase in mass trans-
fer coefficient could be attributed to both increase in diffusion
coefficient and decrease in viscosity [37]. This could probably
explain the higher influence of temperature on mass transfer
coefficient than that on diffusion coefficient. Hence, the mass trans-
fer Biot number increased with temperature. The higher values of
Biot number (>50), indicates the external resistances for mass trans-
fer is negligible confirming efficient mixing between solute and
solvent [45] and therefore, internal transfer is rate-limiting [39].
3.4. Thermodynamics of MAE
The maximum flavonoid content determined using exhaustive
extraction with multiple solvents was found to be 30.76 mg/g,
which is in agreement with flavonoid content in T. bellerica
reported earlier [31]. The thermodynamic parameters of MAE of
flavonoids from T. bellerica are summarized in Table 2. The positive
value of the change in enthalpy (DH°) indicates that the MAE of
Fig. 9. Suitability of diffusion model for MAE of flavonoids from T. bellerica.
175
Table 1
Effective diffusion coefficient, mass transfer coefficient and mass transfer Biot number
of MAE of flavonoids from T. bellerica.
S (mL/g) T (°C) De  1012 (m2/s) KT  105 (m/s) Bi
40 40 5.4387 ± 0.11 1.4610 ± 0.13 170.6334 ± 15.5
60 5.6048 ± 0.21 2.0139 ± 0.19 223.6867 ± 19.5
80 5.7122 ± 0.11 2.8334 ± 0.12 314.5975 ± 13.6
100 5.8368 ± 0.14 4.0216 ± 0.09 443.2038 ± 14.2
flavonoids from T. bellerica is endothermic. The positive value of
the change in entropy (DS°) indicates the increase in disorder of
the solid–liquid extraction system due to the solute transfer from
more ordered solid phase to the less ordered liquid phase [30].
The negative values of change in Gibbs free energy (DG°) indicate
the extraction process to be spontaneous. The thermodynamic
parameters for extraction of flavonoids from T. bellerica have not
been reported earlier and hence the direct comparison with litera-
ture data could not be carried out. However, the values of DH°, DS°
and DG° obtained in the present work are comparable with those
reported for MAE of anthocyanin from grape peel [46]. The equilib-
rium constant in MAE is higher than that in CE (Table 3). This indi-
cates that the solute transport from the solid phase to liquid phase
is more favourable in MAE than that in CE. In addition, the change
in entropy is higher in MAE (Table 2) in comparison with CE
(Table 3). Hence MAE is more feasible than CE, as evident from
changes in Gibbs free energy for MAE and CE.
3.5. Chemical composition of T. bellerica fruits
The composition of microwave assisted aqueous extract of
T. bellerica fruits prepared at 100 °C using the solvent-to-feed ratio
of 40 mL/g and analysed using GC–MS is given in Table 4. The com-
ponent with highest percentage peak area was 1,2,3-benzenetriol.
The flavonoid fraction 4H-pyran-4-one, 2,3-dihydro-3,5-
dihydroxy-6-methyl- was observed to present in a significant
amount. The composition of aqueous extracts of T. bellerica fruit
obtained by CE is compared with that of aqueous extract prepared
by MAE under identical conditions (T = 100 °C, S = 40 mL/g) in
Table 4. It is observed that the major compounds separated using
176 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178

Table 2 superior to conventional extraction with respect to saturation yield

Thermodynamic parameters for MAE of flavonoids from T. bellerica. of flavonoids.
T (K) Ke DH° (kJ/mol) DS° (J/mol K) DG° (kJ/mol)
313 1.8420 15.5488 54.4151
1.4830 3.6. Morphology of plant material after extraction
333 2.3965
2.5714
353 3.3987
3.6597
373 4.7960
4.7480 Scanning electron micrograph of the raw material (Fig. 11a)
indicates that the particle surfaces were planar with few surface

Table 3
Thermodynamic parameters for CE of flavonoids from T. bellerica.

T (K) Ke DH° (kJ/mol) DS° (J/mol K) DG° (kJ/mol)

313 1.2473 10.3900 34.7991
0.5021
333 1.4609
1.1981
353 1.9422
1.8940
373 2.3212
2.5900

Table 4
Composition of aqueous extracts of T. bellerica fruits obtained by MAE and CE.

Compounds % Peak area in

extract prepared
using
MAE CE
Furfural 0.9009 0.8928
3H-pyrazol-3-one, 2,4-dihydro-2,4,5-trimethyl- 0.9565 1.7281
4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- 2.473 2.3276
2-Furancarboxaldehyde, 5-(hydroxymethyl)- 7.7726 7.6983
1,2,3-Benzenetriol 84.1598 79.7393
D-allose 0.7299 0.4183
Other minor compounds 3.0073 7.1956

MAE of T. bellerica fruits were similar to that of obtained by CE [47]

implying there wasn’t any destruction of bioactive compounds due
to microwave exposure. The antioxidant activities (% inhibition) of
aqueous extracts of T. bellerica prepared using CE and MAE at the
solvent-to-feed ratio of 40 mL/g at 100 °C are comparable
(Fig. 10), confirming that there microwave exposure did not cause
the destruction of bioactive compounds.
The saturation flavonoid yield obtained by MAE of T. bellerica
fruits at solvent-to-feed ratio of 40 mL/g and temperature 100 °C
was 25.21 mgQE/g using water as the solvent. Under the same
conditions of solvent-to-feed ratio and temperature, saturation
flavonoid yield of 19.16 mgQE/g was obtained by conventional
extraction using water as the solvent. This shows that under
identical operating conditions microwave assisted extraction is

Fig. 11. Scanning electron micrographs of T. bellerica fruits (a) untreated, (b) after
Fig. 10. Antioxidant activity of aqueous extract of T. bellerica fruits. CE and (c) after MAE.
 R. Yedhu Krishnan, K.S. Rajan / Separation and Purification Technology 157 (2016) 169–178
voids. The scanning electron micrograph of the material after CE
(Fig. 11b) is similar to that of raw material (Fig. 11a), implying that
there were no appreciable changes in the microstructure of the
material during conventional extraction. Hence, the solute–solvent
contact during conventional extraction was predominantly depen-
dent on the microstructure of the raw material. However, the scan-
ning electron micrograph of the plant material after MAE (Fig. 11c)
reveals the presence of a number of long cracks distributed across
the entire particle. The presence of such cracks could be attributed
to absorption of microwave energy by the cellular water, thereby
causing ruptures/cracks in the plant material. It is believed that
these wide openings of the cellular pores would have increased
the surface area for particle–solvent contact and enhanced the
exposure of solute to the solvent. The rapid vaporization results
in dehydration and change in surface tension of the cell wall that
could also have contributed to crumbling of the surface [48,49].
The difference in the scanning electron micrographs of material
after CE and MAE summarily indicates that the microwave expo-
sure has resulted in the increase of solute–solvent contact through
partial destruction of the solid phase and creation of cracks.
4. Conclusion
In the present work, appropriate operating conditions were
identified for MAE of flavonoids from T. bellerica fruits through
studies on the kinetics of extraction. Both solvent-to-feed ratio
and temperature played pivotal role in influencing both kinetic
parameters and the flavonoid yield. T. bellerica was observed to
be a good source of flavonoids providing a maximum yield of
25.21 mg/g using MAE carried out at 40 mL/g and 100 °C. Accord-
ingly, 82.74% of total flavonoids could be recovered by MAE using
water as the solvent, while only 63.75% could be recovered by CE.
The thermodynamic parameters revealed the MAE process to be
endothermic and spontaneous. The GC–MS data showed that
the primary compounds in the extracts obtained by MAE were
same as those obtained using conventional extraction indicating
the negligible influence of microwave irradiation on the stability
of compounds. The kinetic model represented as a function of
temperature and solvent-to-feed-ratio can be used to determine
the residence time required to achieve a desired flavonoid
concentration in the extract in a continuous MAE of flavonoids
from T. bellerica using water as the solvent.
Acknowledgements
This work is supported by (i) Grant No. SR/FST/ETI-331/2013,
Department of Science & Technology (DST), India (ii) SRF vide.
09/1095/0007/2014-EMR-I, Council of Scientific & Industrial
Research (CSIR), India (iii) SASTRA University. The authors thank
Dr. V. Vadivel and Dr. S. Sriram, CARISM, SASTRA University for
their help in carrying out the DPPH assay.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.seppur.2015.11.
035.
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