Chemical Engineering Communications

ISSN: 0098-6445 (Print) 1563-5201 (Online) Journal homepage: http://www.tandfonline.com/loi/gcec20

Microwave Assisted Batch Extraction of

Polyphenols From Sea Buckthorn Leaves
Ioana Asofiei, Ioan Calinescu, Adrian Trifan, Iulia Gabriela David & Adina
Ionuta Gavrila
To cite this article: Ioana Asofiei, Ioan Calinescu, Adrian Trifan, Iulia Gabriela David & Adina
Ionuta Gavrila (2016): Microwave Assisted Batch Extraction of Polyphenols From Sea Buckthorn
Leaves, Chemical Engineering Communications, DOI: 10.1080/00986445.2015.1134518
To link to this article: http://dx.doi.org/10.1080/00986445.2015.1134518

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Date: 27 March 2016, At: 23:56

Microwave Assisted Batch Extraction of Polyphenols from Sea Buckthorn Leaves

Ioana Asofiei1, Ioan Calinescu1, Adrian Trifan1, Iulia Gabriela David2, Adina Ionuta
Gavrila1
1

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Department of Bioresources and Polymer Science, University Politehnica of Bucharest,

1-7 Gh. Polizu, Zip Code 011061, Bucharest, Romania, 2Department of Analytical
Chemistry, University of Bucharest, 90 Panduri, Zip Code 050657, Bucharest, Romania

Corresponding author to Adina Ionuta Gavrila, Department of Bioresources and Polymer

Science, University Politehnica of Bucharest, 1-7 Gh. Polizu, Zip Code 011061,
Bucharest, Romania. E-mail: adinagav@yahoo.com

Abstract
Extraction of polyphenols from Sea Buckthorn leaves using microwave assisted
extraction (MAE) is described. The influence of different parameters on the extraction
process (reactor type, stirring rate, extraction time, temperature, ethanol/water ratio) was
studied. The polyphenolic extracts were analyzed in order to determine the total phenolic
content (TPC) by Folin-Ciocalteu method or by differential pulse voltammetry (DPV)
and the concentration of the main polyphenolic compounds by HPLC. The specific
microwave energy was also determined. MAE leads to a shorter extraction time (7.5 min
versus 30 min for the conventional method). The best results of MAE were obtained at a
temperature of 90C, using a solvent/plant ratio of 20/1 and 50% ethanol in the extraction
solvent. The highest values of antioxidant capacity were obtained for polyphenolic
extracts resulted from microwave extraction.

KEYWORDS: microwave assisted extraction, polyphenols, Sea Buckthorn

INTRODUCTION

Hippophae rhamnoides L. known as Sea Buckthorn is a nitrogen fixing bush widely

found in temperate and subtropical zones. All parts of the shrub are rich in bioactive
compounds. Recent developments have shown the beneficial properties of Sea Buckthorn
leaves extracts. The leaves contain polyphenolic compounds such as ferulic acid, caffeic

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acid, flavonols, gallic acid, kaempferol, isorhamnetin, epigallocatechin, epicatechin, and

gallocatechin. The high content of polyphenols in plant leaves have shown antioxidant
properties and can be used to prevent the damaging effect of oxidant radicals
(Upadhyayet al., 2010; Kant et al., 2012). These polyphenols present pharmacological
activities such as radioprotective, immunomodulatory, anti-inflammatory and tissue
regeneration (Chawla et al., 2007; Suryakumaret al., 2011).

Extraction of bioactive compounds from vegetable materials is determined by different

factors such as extraction yield, production cost and uses of safety and environmental
friendly solvents (Desai et al., 2010). The most common extraction methods are Soxhlet
extraction, conventional extraction and heat reflux extraction. These methods require
long extraction times, expensive organic solvents, relatively high solvent consumption
and high temperatures that could cause the degradation of valuable constituents.

Recent developments present more advantageous techniques such as pressurized fluid

extraction (PFE), ultrasound assisted extraction (UAE), pulsed electric field (PEF),
supercritical fluid extraction (SFE) microwave hydro-diffusion and gravity (MHG) and
microwave assisted extraction (MAE).

Extraction of bioactive compounds by PFE technique is carried out under high

temperature and pressure. This method is unhelpful for polyphenols which are heat
sensitive (Kronholm et al., 2007). In UAE of polyphenols from plants the rupture of
matrix is initiated by ultrasonic irradiation. By sonication the solvent penetrates the plant

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cells due to the jet generated by the bubble breakdown. This jet enlarges the cell pores
and act as a micro-pump, forcing the solvent to enter into the cells and dissolve the
components (Mason et al., 2011). Thus, the active components rapidly diffuse from plant
tissue to solvent. UAE method is useful for thermolabile compounds due to the short
extraction time. The extraction efficiency is influenced by the US power which is lower
for longer extraction time (Pasrija et al., 2015).

PEF treatment increases the permeability of plant tissues. The cell membranes are
charged and pores are formed in the membranes facilitating extraction of cellular fluid.
PEF has been used to extract polyphenols. The PEF efficiency was improved when the
treatment was performed in the presence of ethanol. PEF pre-treatment increased both the
extraction kinetics and the maximum yield of polyphenols (Chemat et al., 2015).
The SFE technique using CO2 as a solvent has the advantages of mild extraction
conditions (short extraction time and low extraction temperature), but CO2 is non-polar
and this could limit its use in the extraction of polar polyphenols (Temelli et al., 2009).
MAE is a promising technology to extract bioactive compounds from vegetal material by
combination of extraction technique with microwave heating. The microwave heating is
based on the effect of the microwaves on material molecules by ionic conduction and
dipole rotation. Thus, an efficient heating requires either polar extraction solvents or

polar materials (Desai et al., 2010; Lew et al., 2002). Recently, MAE has been
extensively employed for the extraction of polyphenols from various parts of plants due
to its benefits such as shorter extraction time, good control of heating process, high
extraction efficiency and selectivity, better extraction yield and reduced quantity of

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energy (Prino-Issartier et al., 2010, Chemat et al., 2015, Filly et al., 2014, Li et al., 2013,
Xiao et al., 2008; Rostagno et al., 2007; Chen et al., 2007; Spigno et al., 2009; Wanget
al., 2010; Alupului et al., 2009; Calinescu et al., 2014; Calinescu et al., 2002).

MHG is a green extraction technique that combines microwave heating and gravity at
atmospheric pressure. MHG is a suitable method for laboratory and industrial-scale
applications (extraction of pigments, essential oils, and antioxidants from plants).
Extraction of polyphenolic compounds from onion and Sea Buckthorn by MHG led to a
shorter extraction time, no solvent used, and polyphenolic extract showed a higher
phenolic content with greater antioxidant activity in comparison to classical extraction
methods (Li et al., 2013).

The purpose of this paper is to establish the optimal conditions for polyphenols extraction
from Sea Buckthorn leaves using batch microwave assisted technique. The influence of
different extraction parameters such as reactor type, stirring rate, extraction temperature
and time, solvent to plant material ratio and solvent concentration have been studied.

MATERIALS AND METHODS

A. Materials

The Sea Buckthorn leaves (Hippophae Rhamnoides L.) were harvested in the summer of
2014 at Hofigal S.A. in Furculesti. The fresh leaves were dried in air flow-heating oven at
60C to a constant weight. The dried leaves were ground using an electric grinder and
sieved to a particle size under 0.5 mm. The ground Sea Buckthorn leaves were dosed in

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samples of 25 g (in sealed plastic vessel) and stored at 4-5C until they were used for
extraction of the phenolic compounds.

Folin-Ciocalteu reagent (Merck), ethanol and sodium carbonate were analytical grade.
The standard used for TPC analysis was gallic acid from Sigma-Aldrich. For the HPLC
quantification of phenolic compounds, the following standards were used: gallic acid,
caffeic acid, chlorogenic acid, catechin, ferulic acid, p-coumaric acid and rutin from
Sigma-Aldrich.

B. Polyphenols Extraction Procedure

The MAE of phenolic compounds was performed using a microwave system (Biotage
Initiator). The conventional extraction of polyphenolic compounds was carried out in a
water bath using a heating plate equipped with a temperature controller unit and magnetic
stirring. The extractions were performed in triplicate, using a 20:1 ratio of solvent to
plant. The experiments were carried out at different temperatures (60C, 90C and
120C) and different stirring rates (300 and 900 rpm). The extractions occur in a mixture
of ethanol in water using a concentration of 0%, 25% and 50% ethanol in water.
Individual experiments were performed considering the following extraction times: 50,
100, 150, 200, 300, 450, 600, 900 and 1800s. After the extraction, the mixture was

centrifuged at 3000 rpm for 5 min at room temperature and the supernatant was collected
and fresh analyzed every time.

C. Analysis

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Determination Of Total Phenolic Content

Total phenolic content of extracts was determined colorimetrically using the FolinCiocalteu method according to International Standard ISO 14502-1 with minor
modifications. The fresh extracts were diluted 125 times with distilled water. Further, 0.5
mL of diluted extract was mixed with 5 mL of 10% Folin-Ciocalteu reagent and stirred
for 5 min to perform the reaction. Next, 1.5 mL of 20% Na2CO310H2O and 3 mL of
distilled water were added. Before analysis, the samples were kept for 30 min in the dark
at room temperature. The absorbance was measured at 760 nm using a Shimadzu UV
mini-1240 UV/Visible Scanning Spectrophotometer. The samples were analyzed in
duplicates. The results were quantified as milligram of gallic acid equivalents per 1 gram
of dry matter (mg GAE/g DM) using standard curve prepared for gallic acid solutions (15 mg/mL).

DPV Determination Of Total Phenolic Content

Voltammetric measurements were carried out using an electrochemical system
(potentiostat/galvanostat) AUTOLAB PGSTAT 12. The system was equipped with a
voltammetric cell consisting of a working electrode (pencil graphite electrode - PGE)
(David et al., 2015), an Ag/AgCl (3M KCI) reference electrode and a platinum wire as
auxiliary electrode. The optimized parameters for the differential pulse voltammetric

(DPV) measurements were the following: initial potential + 0.200 V, final potential +
1.000 V, scan rate v=50 mV/s, pulse period PP=180 msec, pulse width PW=60 msec,
sample width SW=10 msec, pulse amplitude PA= E=20 mV.

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Differential pulse voltammograms (DPVs) were recorded for 10 mL of 1000 fold diluted
extract samples. 0.1 M H2SO4 was used as supporting electrolyte. If the potential applied
to the PGE was scaned towards anodic values, the polyphenolic compounds from plant
extracts are oxidized. The DPVs of all samples showed an anodic peak at potentials
higher than 500 mV. The intensity of this peak was proportional to the amount of total
polyphenols contained in the analysed extract. Increasing the dilution leads to a more
pronounced peak. At 1.000 times dilutions, the peak current varied linearly with the gallic
acid (GA) concentration added in the sample as standard.

In order to determine the TPC of extracts the standard addition method was used. Thus,
for each sample DPVs were recorded before and after each of the three additions of 0.1
mL GA stock solution (2.9410-3 M) (Figure 1A). The TPC of samples was calculated
using the equation of the Ipa = f (Cad) curve where Ipa represents the maximum peak
intensity and Cad the concentration of added GA (Figure 1B).

HPLC Analysis Of Major Phenolic Compounds

The polyphenolic compounds extracted were further analyzed by HPLC analysis. The
analyses were undertaken with the help of a Jasco HPLC. The system includes: UV-2075
detector; PU-2080 plus pump; LG-2080_4 gradient unit; DG-2080_4 degasser;

Teknokroma Nucleosil 100 C18 (10micro m, 250x0.4) separation column. Analyses were
performed at a flow rate of 0.5 mL/min using water with 2% v/v acetic acid (solvent A)
and methanol (solvent B) under the following gradient program: 0-9 min 70% A and 30%
B, 9-18 min 60% A and 40% B, 18-30 min 50% A and 50% B, 30-60 min 50% A and

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50% B and then returned to initial condition for a 10 min reequilibration, with total run
time 70 min. The analytes were detected at 270 nm. For better separation of polyphenols
they were extracted from the ethanolic solution resulted from extraction. Extraction of
polyphenols from ethanolic solvent was performed using ethyl ether as solvent (50%
polyphenols solution and 50% ethyl ether are stirred 10 min at room temperature).
Samples of polyphenols in ethyl ether have been used for HPLC analysis. Analytes were
identified by comparison of retention times with known standards.

RESULTS AND DISCUSSION

1. Factors That Influence The MAE Of Polyphenolic Compounds From Sea
Buckthorn Leaves
A) Influence Of The Stirring Rate On The Microwave Polyphenols Extraction
Polyphenols extraction from Sea Buckthorn leaves is a complex process influenced by a
very good contact between solvent and plant material. Therefore, in order to allow the
diffusion of the solvent in plant tissue and an efficient extraction of bioactive compounds,
intensely mixture stirring is required. One of the studied parameters was the influence of
stirring rate on the extraction of polyphenolic compounds. Extraction of polyphenols was
carried out at different stirring rates (300 and 900 rpm) and different extraction times.
The results are shown in Figure 2.

As shown in figure 2, the increase of the stirring rate, from 300 rpm to 900 rpm, led to a
three fold increase in the total phenolic content. In addition, at a stirring rate of 300 rpm,
a longer extraction time determines a slow increase of TPC. For this reason, a low stirring
rate requires very long extraction times in order to achieve an efficient extraction.

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Moreover, it is noted that for higher stirring rate (900 rpm) a longer extraction time (over
200 s) leads to a slight decrease in TPC.

B) Influence Of The Reactor Type On The Microwave Batch Extraction Of

Polyphenols
The extraction process is more effective if the stirring rate is higher. A method of
improving the mixing of the extraction medium is to change the geometry of the reactor.
Therefore, the reactor was modified in order to allow the use of a bigger magnetic stirrer.
For this study, extraction of polyphenolic compounds was carried out using two types of
reactors (normal Biotage reactor - 1 and a modified reactor 2, as shown in Figure S1).
The two reactors make possible the extraction of polyphenols using the same volume of
solvent (5 mL). Thus, the microwave extractions were performed at various stirring rates
using these two types of reactors. The results of the influence of the reactor type on the
polyphenols extraction are shown in Figure 3.

Changing the geometry of the reactor and using a larger stirrer improved the extraction
process of polyphenols, as shown in Figure 3. Although, at short extraction times, the
polyphenols concentration is about the same for both reactors, at longer times (after 200
s) the TPC is significantly higher (around 20%) for modified reactor (Fig. S1, reactor 2).

Given the fact that the highest amounts of polyphenols were obtained for reactor 2, the
following studies of polyphenols extraction from Sea Buckthorn leaves were carried out
using this reactor.

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C) Influence Of Ethanol Concentration On The Extraction Of Polyphenols

The most common method of extraction of polyphenols from plants involves polar
organic solvents that destroy the cell membrane and dissolve the polyphenols. Most
organic solvents are volatile, flammable, and often toxic and are responsible for the
greenhouse effect and environmental pollution. The increasing demand of green
extraction, using natural products and extracts, made us use a less toxic polar bio-solvent
as ethanol. Even if it is potentially explosive and inflammable, ethanol is used on a large
scale due to its low price, biodegradability and availability in high purity (Chemat et al.,
2012). This solvent is considered a natural solvent (Chemat et al., 2015) that can be
produced from agricultural sources sugar beet and cereals. The study of the effect of
solvent on the extraction process was achieved by using different concentrations of
ethanol in water (Figure 4).

The increase of the concentration of ethanol in the extraction solvent mixture increases
the TPC, as shown in Figure 4. Thus, the use of a 25% ethanol concentration in water
results in achieving polyphenol concentrations of about 90 mgGAE/g DM, higher than
when using only water as solvent. Moreover, increasing the concentration of ethanol in
water to 50% doubles the amount of extracted polyphenols compared to the extraction
without ethanol. This behavior can be explained by the difference in solubility of

10

compounds in water and ethanol. The organic solvent (ethanol) dissolves cell membranes
and water causes the swelling of the vegetable material, thus allowing the solvent to
penetrate more easily into the solid matrix. Thereby, the extractability of polyphenols
increases. Therefore, the choice of the solvent / water ratio depends on the composition of

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the polyphenols. In conclusion, a concentration of 50% ethanol in water results in an

efficient extraction of polyphenols from Sea Buckthorn leaves at a temperature of 60 C.

D) Influence Of Temperature And Extraction Time On The Polyphenols Extraction

Another studied parameter on polyphenols extraction from Sea Buckthorn leaves is the
effect of temperature on the TPC. The temperature is an important factor in the solidliquid extraction process. The increase of the temperature increases the solubility of the
polyphenols and also increases the mass-transfer coefficient between the matrix material
and extraction medium. Thus, the experiments were carried out at different temperatures.
The extraction of phenolic compounds is also influenced by the extraction time, which
can affect the solubility of polyphenols. A long extraction time can lead to the oxidation
of polyphenols or can change the conformation of extracted polyphenols.

The microwave power is an important factor for microwave assisted extraction of

bioactive compounds from plants. For each experiment, the microwave power and
temperature profiles have been recorded. Using the above mentioned recorded values, we
determined average microwave power during heating and extraction, and the microwave
energy supplied to the system (Equations S1-S7, Table SI). The specific energy was
determined by the following equation:

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Esp

Etotal
TPC

J
mgGAE

(1)

Where: Esp - specific energy [J/mg GAE]; Etotal total energy [J]; TPC total phenolic
content [mg GAE/g DM].

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Figure 5 shows the influence of extraction time and temperature on the TPC and specific
energy by batch MAE from Sea Buckthorn leaves.

A higher temperature increases the polyphenols content. This can be explained by the
increase of polyphenols solubility and a high value of mass-transfer coefficient between
the matrix and the extraction medium at high temperatures. In addition, a high extraction
temperature results in the destruction of the plant cell walls. Thus, this can lead to a
further better penetration of the solvent in the cell walls, thus allowing the dissolution of
polyphenols in the extract. Likewise, Figure 5 shows that at high temperature (120C) the
maximum TPC is obtained faster than for lower temperatures (100 s for 120C and 450 s
for 60C and 90C). However, the highest concentration of polyphenols was obtained for
the extraction temperature of 90C. After exceeding the maximum, at a temperature of
120 C the TPC decreases much faster than the extractions at 60 C or 90 C. This can
be explained by the degradation of polyphenols at high temperatures. From these data it
was concluded that the optimal temperature for MAE of polyphenols from Sea Buckthorn
leaves is 90C.

As is shown in figure 5, the lowest specific energy corresponds to the experiments carried
out at 60 C. At 90 C, slightly higher values of specific energy are obtained. In addition,
12

the amount of extracted polyphenols at 90 C is higher than the amount obtained for
other extractions (60 C and 120 C). For this reason, the extraction at 90 C can be
considered more efficient from TPC and specific energy points of view.

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These values of specific energy are expected to decrease when scaling up. This effect is
typical for pilot and industrial installations where a higher ratio between the microwave
energy absorbed by the system and the one delivered is required. Reactors with small
volumes are used in the experiments presented in this work and for this reason, the ratio
between the required and total energy is only 1 to 4 (Table SI).

2. Microwave Assisted Extraction Vs. Conventional Extraction

To highlight the effect of microwaves on polyphenols extraction from Sea Buckthorn
leaves, experiments were carried out under the same conditions, for various temperatures,
both by microwave assisted extraction and conventional extraction (Figure 6).
The increase of the extraction time leads to an increase in the total phenolic content,
reaching a maximum after 7.5 min in the MAE and after 30 min in the case of
conventional extraction. However, a further increase of the extraction time leads to the
decrease of the polyphenolic content. Therefore, although the maximum concentration of
polyphenols is about the same for both methods, the MAE leads to reducing the
extraction time.

3. Total Phenolic Content Determination By Differential Pulse Voltammetry

13

Differential Pulse Voltammetry (DPV) can measure the ability of compounds to donate
electrons. This can be related to the antioxidant capacity of polyphenolic compounds. For
lower values of the oxidation peak potential, the oxidation occurs easily. Therefore, the

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antioxidant capacity of the compound is higher (Abou Samra et al., 2011).

Table I presents a comparison between the results of the TPC analysis performed by the
Folin-Ciocalteu method and by DPV. One can observe that the DPV values are lower
than those obtained applying the Folin-Ciocalteu method. This can be explained by the
fact that the Folin-Ciocalteu reagent is less selective and can be also reduced by other
non-phenolic compounds present in the analysed samples (Seruga et al., 2011).

4. Polyphenols Composition
TPC values can be influenced by a number of other compounds with reducing character
that are extracted from plant (as shown by comparing TPC values obtained by Folin
Ciocalteu and DPV methods). For this reason, the polyphenolic extracts were analyzed by
HPLC analysis to quantify the content of polyphenols (Table II). The phenolic
compounds analyzed by HPLC depended on the extraction method. The main
components from Sea Buckthorn leaves extracts were gallic acid, catechin, caffeic acid
and rutin (Bittova et al., 2014). Catechin was the predominant polyphenolic species,
followed by gallic acid and rutin.

14

From the results shown in Table II it can be noticed that the increase of extraction time
led to the increase of total phenolic content (TPC) values, but the major components
content slightly decreased at long extraction time.

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CONCLUSIONS
The aim of this work was to determine the best conditions for MAE of polyphenols from
Sea Buckthorn leaves. The influence of various parameters was determined: stirring rate,
extraction time, type of reactor, concentration of ethanol in water and temperature. MAE
leads to reduction of the extraction time compared to conventional extraction. Increasing
the temperature leads to higher TPC, but at long extraction time concentration of
polyphenols decreases. This can be explained by the degradation of polyphenols. A
higher stirring rate increases the polyphenols content, but another important factor in
order to obtain an efficient stirring of the extraction medium is the geometry of the
reactor. Total phenolic content increases with the increase of the concentration of ethanol
in water. However, the ratio of the solvent is closely related with the solubility of
polyphenolic compounds found in Sea Buckthorn leaves.

The effect of heating time and specific energy on the total polyphenolic content was
investigated. In comparison with other extraction temperatures, the experiments carried
out at 90 C led to a higher TPC obtained with a lower value of specific energy.

DPV analysis was used as an alternative method for TPC determination. The values
obtained by DPV are lower than those resulted by Folin-Ciocalteu method, due to the fact

15

that Folin-Ciocalteu reagent is less selective and can be also reduced by other nonphenolic compounds. This low selectivity was confirmed by the HPLC analysis.

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Table 1.

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No.

1
2
3
4
5

Experimental conditions
Ethanol
concentration,
[%]
50
25
0
0
50

Extraction
time, [s]
450
450
450
150
450

TPC [mg
GAE/g DM]
Heating
FolinDPV
type
Ciocalteu
method
method
Microwave
134.96
93.71
Microwave
95.02
70.34
Microwave
66.00
47.90
Microwave
62.13
41.40
Conventional 123.93
86.49

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Table 2.

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No. Temperature, Time,

Compound [mg/g DM]
[C]
[s]
Gallic Catechin Caffeic Rutin
acid
acid
1.
2.
3.
4.

90
90
90
90

50
100
200
300

3.24
3.34
3.31
3.13

9.37
10.09
9.24
9.02

21

1.51
1.64
1.44
1.4

3.94
3.79
4.02
3.01

TPC,
[mg
GAE/g
DM]
109.75
114.84
125.31
131.14

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Figure 1.

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Downloaded by [Anelis Plus Consortium 2015], [Adina Gavrila] at 23:56 27 March 2016

Figure 2.

23

Downloaded by [Anelis Plus Consortium 2015], [Adina Gavrila] at 23:56 27 March 2016

Figure 3.

24

Downloaded by [Anelis Plus Consortium 2015], [Adina Gavrila] at 23:56 27 March 2016

Figure 4.

25

Downloaded by [Anelis Plus Consortium 2015], [Adina Gavrila] at 23:56 27 March 2016

Figure 5.

26

Downloaded by [Anelis Plus Consortium 2015], [Adina Gavrila] at 23:56 27 March 2016

Figure 6.

27