IIUM Engineering Journal, Vol. 18, No. 2, 2017
Mohd Said and Quan
RECOVERY OF THE BIOLOGICAL ACTIVE
COMPOUNDS OF Musa Sp. THROUGH MICROWAVE
ASSISTED EXTRACTION
FARHAN MOHD SAID* AND TAN KAI QUAN
Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang,
Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia.
*Corresponding author: farhan@ump.edu.my
(Received: 20th Feb. 2017; Accepted: 31st Oct. 2017; Published on-line: 1st Dec. 2017)
ABSTRACT: This study set out to investigate the recovery of the biological active
compounds of Musa sp. through microwave-assisted extraction (MAE) system. The aim
of this paper is to critically analyze the effects of temperature, microwave power,
irradiation time, and solid to liquid ratio on antioxidant activity and phenolic compounds.
The extraction was conducted on the unripe peel, unripe pulp, ripe peel, and ripe pulp of
Musa sp. The extraction process was carried out by utilizing distilled water as an
extracting agent. The antioxidant activity and phenolic compounds for unripe and ripe
Musa sp were observed to be extracted best at 70 oC, microwave power range of 500 W
– 800 W, and 90 s irradiation time at 3:60 solid to liquid ratio. Overall, antioxidant and
phenolic compounds were found to be significantly higher in the following order: unripe
peel, ripe peel, unripe pulp, and ripe pulp.
ABSTRAK: Kajian ini adalah untuk mengkaji kedapatan sebatian aktif biologi dari Musa
sp. mengguna sistem ekstrak mikrogelombang (MAE). Objektif kajian ini adalah untuk
menganalisa dengan kritikal kesan suhu, kuasa gelombang mikro, tempoh sinaran
gelombang dan nisbah pepejal kepada cecair terhadap aktiviti antioksida dan sebatian
fenolik. Pengekstrakan dilakukan pada kulit yang belum matang, pulpa yang belum
matang, kulit masak dan pulpa masak Musa sp. Proses pengekstrakan dilakukan dengan
menggunakan air suling sebagai agen pengekstrak. Aktiviti antioksidan dan sebatian
fenolik bagi Musa sp. yang masak dan tidak masak adalah paling baik diekstrak pada
suhu 70 oC, kuasa gelombang mikro antara 500 W – 800 W, tempoh iradiasi selama 90
saat pada nisbah 3:60 pepejal kepada cecair. Keseluruhannya, sebatian antioksidan dan
fenolik adalah lebih tinggi pada turutan berikut: kulit yang belum matang, kulit masak,
pulpa yang belum matang dan pulpa masak.
KEYWORDS: antioxidant; phenolic; microwave assisted extraction; Musa sp.; toxicity free
solvent
1. INTRODUCTION
Generally, Musa sp. is known as banana, and it is one of the most consumed
worldwide fruits [1] and has also been classified as one of the antioxidative foods [2].
Results showed that Musa sp. pulp and peel contain various antioxidants such as vitamins,
β-carotene as well as phenolic compounds such as catechin, epicatechin, lignin and
tannins, including anthocyanins [3-5]. In addition, Musa sp. is also remarkably rich with
minerals such as potassium and phosphorus [4, 6]. The peels possess higher phenolic
compounds and antioxidant properties [3, 7] along with mineral contents compared to
Musa sp. pulps. [8].
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Antioxidants can be found either in plant materials or supplements (synthetic). There
are zero side effects that have been reported on the use of natural antioxidants from plants,
whereas synthetic antioxidants were found to have a genotoxic effect [9]. In addition,
natural antioxidant agents have grabbed serious attention due to their ability to scavenge
free radicals [10]. There is crucial interest on free radicals due to the development of a
number of disorders, including cancer, neurodegeneration, and inflammation [11]. The
presence of antioxidants such as phenolics and flavonoids in plants may provide protection
against a number of diseases, in which the mortality from degenerative disorders has
inversely led to the ingestion of natural antioxidants [12]. Hence, its application on foods
allows rancidity to be minimized as well as retard the formation of toxic oxidation
products, which helps maintain the nutritional quality and increase food shelf life [13].
Natural antioxidants are in high demand, especially to be applied in nutraceuticals,
biopharmaceuticals, and food additive pertinent to consumer preferences. In recent years,
apart from an antioxidant, phenolic compounds have also received much attention as a
result of their potential ability and beneficial implications in human health [14].
Several studies have identified the extraction of antioxidants and phenolic
compounds. Direct extraction using solvents is the most common technique employed to
obtain extracts with high antioxidant activity. An extraction process is intended to achieve
the maximum yield of substances and the highest quality of targeted compounds [15]. The
solvent extraction has been widely used to extract bioactive components from the plants
[16]. The standard solvents for extracting antioxidant include methanol, ethanol, and
acetone, which are used either separately or in combination with an aqueous solution [5,
17-19]. For the past 10 to 15 years, the interest in MAE has significantly increased in
regards of their advantages which include its ability to reduce the extraction time, solvent
volume, and improve extraction yield [20-23] in comparison to the traditional extraction
techniques (e.g. Soxhlet extraction and hot water extraction (HWE)). It is important to
note that conventional extraction methods have been associated with high solvent
requirements, longer extraction times and a higher risk of degradation of heat sensitive
constituents. In addition, MAE is a green technology and considered as a potential
alternative to the conventional solid-liquid extraction of bioactive compounds from plant
matrices [24].
Furthermore, MAE is a process that uses microwave energy and solvents to extract
the targeted compounds from the matrices. Microwave energy acts as a non-ionising
radiation that causes rotation of the dipoles. The extremely high temperature at the
restricted area of MAE was found to cause selective migration of target compounds from
material to the surroundings at a higher rapid rate. MAE performs similar or better
recoveries compared to the conventional extraction method. In MAE, the solvent and
sample are contained in sealed extraction vessels under controlled temperature and
pressure conditions. The closed vessels allow the temperature of the solvent to increase
above its boiling point, which then shortens the extraction time and subsequently increases
extraction efficiency. The efficiency of MAE depends on several variables which may not
be suitable for all plant materials as a result of different nature of the existing bioactive
phytochemicals [22, 25].
It has been observed that up to this date, there is no report on the use of a toxicity-free
solvent in MAE on the maturity pulp and peel of Musa sp. Therefore, the specific
objective of the study was to determine the effect of MAE method using distilled water as
a toxicity free solvent in extracting antioxidant and phenolic compounds of ripe and unripe
Musa sp. pulp and peel.
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2. MATERIALS AND METHOD
2.1 Chemicals
Methanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), gallic acid (GA), Folin-Ciocalteu
reagent, sodium bicarbonate was obtained from Sigma-Aldrich.
2.2 Sample Material and Preparation
The Musa sp. was obtained from a local supermarket at Kuantan, Pahang, Malaysia.
An approximate of 2 kg unripe (green) and ripe Musa sp. (yellow) were washed and
separated into pulps and peels, which was then sliced into a thickness of 2 mm. Musa sp.
sliced sample was dried overnight at 60 oC using an oven prior to the extraction [26].
Meanwhile, fresh Musa sp. sample was prepared freshly.
2.3 Microwave-assisted Extraction (MAE)
The extractions of antioxidant activity and total phenolic content were performed
using MAE (Brand Ethos E touch control, Milestone Corporation, Monroe, CT). A total of
30 g fresh and dried pulp Musa sp. was investigated at different extraction temperature (40
o
C to 70 oC). Other parameters such as microwave power, distilled water, and irradiation
time were fixed at 800 W, 600 ml, and 60 s, respectively.
A series of the experiment was conducted by manipulating other parameters which
include microwave power (100 W to 800 W), irradiation time (30 s to 90 s), and solid to
liquid ratio (1:60 to 3:60). The temperature was fixed at 70 oC. Fresh Musa sp. was
examined during the experiment. On top of that, fresh Musa sp. was divided into 4 types
throughout the process, namely unripe peel, unripe pulp, ripe peel, and ripe pulp. All
samples were performed in triplicate.
2.4 Antioxidant Activity using Free Radical (DPPH)
This method requires 0.2 nM solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in
methanol to be prepared. Next, 0.5 ml of the extract was added to 3 ml of methanol,
followed by 0.3 ml of DPPH added in methanol. The mixture was known as the sample
extract. The blank sample contains 3.3 ml of methanol and 0.5 ml of extract, while control
sample contains 3.5 ml of methanol and 0.3 ml of DPPH in methanol. The mixture was
shaken immediately and left standing at room temperature. This experiment was
conducted in the dark. The mixture was then measured in a UV-VIS of absorbance at 517
nm after 30 min [27]. The antioxidant activity was measured by taking into account the
inhibition of free radical (%) as shown in Eq. (1). Samples were analyzed in triplicate and
average values were calculated.
Inhibition of free radical (DPPH)(%)
Ao A
100%
Ao
(1)
Ao refers to the absorbance of DPPH solution without a sample, while A represents
the absorbance of the test sample mixed with DPPH solution.
2.5 Total Phenolics Content Determination
The concentration of total phenolic compounds was determined using the Folin–
Ciocalteu total phenol procedure with certain modifications as described by Spanos and
Wrolstad [28]. Gallic acid (GA) standard solutions were prepared at 0.0 to 0.5 mg/ml. The
extracts (0.1 ml) and the GA standards (0.1 ml) were transferred into 15 ml test tubes. 3.0
ml of 0.2 N Folin–Ciocalteu reagent were added to each test tube and mixed using a vortex
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mixer. After 1 minute, 2.0 ml of 9.0% (w/v) sodium carbonate (Na2CO3) in water were
added and mixed. The absorbance at 765 nm was determined using a UV-VIS
spectrophotometer (Hitachi U-1800) after being left for 2 h at room temperature [29]. All
samples were determined in triplicate. Total phenolic content (TPC) was expressed as mg
GA per g fresh weight (FW) Musa sp.
3. RESULTS AND DISCUSSION
3.1 Effect of Fresh and Dried Pulp on the Temperature of % Scavenging Activity
The purpose of preliminary studies is to determine the antioxidant activity of fresh
and dried pulp Musa sp. at different temperatures. Other parameters such as the ratio of
solid to liquid, microwave power, and irradiation time were also fixed. The antioxidant
activity was determined using methanol solution of DPPH reagent. The result showed that
freshly prepared DPPH solution fades the deep purple color when antioxidant molecules
extinguish DPPH free radicals and change them into a colorless or bleached product, thus
resulting the absorbance to be reduced at 517 nm band [30]. As can be seen in Figure 1,
the antioxidant activity is expressed in terms of percentage of inhibition of free radical
(%). According to the same figure, both fresh and dried pulps show the highest antioxidant
activity when the temperature is set at 70 oC. The antioxidant activity concentration shows
major increment when the microwave temperature is increased from 40 oC to 70 oC. At 70
o
C, the antioxidant activity of fresh pulp is 45% higher compared to the dried pulps
represented by 58% and 40%, respectively. The dehydration of the samples caused a
decrease of the antioxidant to about 50% on average. The difference of antioxidant activity
might be caused by the process of drying performed on the dried pulp, which resulted in
the breakdown of some of the antioxidant compound and denatured at high temperature (>
70 oC) or evaporated during the drying process. These results match those observed in
earlier studies where antioxidants are heat sensitive, whereby prolong heat treatment may
cause irreversible chemical changes to the antioxidant contents [31-33].
Fig. 1: Antioxidant activity of fresh and dried pulp Musa sp at different temperatures.
3.2 Effect of Microwave Power on Antioxidant Activity and Total Phenolic Content
The effect of microwave power was investigated on the antioxidant activity and total
phenolic compounds of pulp and peel of ripe and unripe Musa sp. Results obtained for
each element are illustrated in Fig. 2 and Fig. 3, respectively. Generally, the power applied
in MAE was found to be significantly affecting the antioxidant activity of ripe and unripe
Musa sp., pulp and peel (Fig. 2). As shown in Fig. 2, the trend of antioxidant activities of
ripe and unripe pulp and peel Musa sp. is dissimilar. Meanwhile, the antioxidant activity
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observed on ripe pulp is significantly higher at 500 W with 82% inhibition of free radical.
The result is different for 800 W and 100 W, where the inhibition of free radicals is 52.1%
and 52.4%, respectively (Fig. 2a). On the other hand, for the ripe peel, no significant
difference of antioxidant activity is observed for both microwave power 500 W or 800 W
(Fig. 2a).
(a)
(b)
Fig. 2: Antioxidant activity of ripe pulp and peel (a) and unripe pulp and peel
(b) at a different microwave power of MAE. Temperature, irradiation time
and solid to liquid ratio were fixed at 70 oC, 60 s and 3:60, respectively.
On the other hand, the antioxidant activities were increased with the escalation of
MAE power from 100 W to 800 W, for both the unripe pulp and peel. According to the
results, the extract obtained from the peel contains higher antioxidant activity compared to
the pulp, especially for the unripe Musa sp (Fig. 2b). This result is consistent with
Fatemah et al. suggestion [34]. The increase of the microwave power of MAE resulted in
higher antioxidant activity, whereby the inhibition of free radical is 65% and 91% on
unripe pulp and peel at 800 W, respectively (Fig. 2b). The increased in the antioxidant
activity was due to the increase of microwave power in MAE. This may be associated with
the direct effects of microwave energy on biomolecules through ionic conduction and
dipole rotation. The microwave energy tends to cause the power to be dissipated inside the
solvent and plant material, which later generates molecular movement and heating. As a
result, the cell walls may be ruptured, which enhances the extraction of antioxidant
activity [35-37]. In addition, more electromagnetic energy was transferred to the extraction
system, which also improved the extraction efficiency when the microwave power is
increased from 100 W to 800 W. However, this phenomenon was not the case for ripe
Musa sp. (Fig. 2a) that obtained higher antioxidant on the pulp. This might be caused by
other compounds other than phenolics and flavonoids that were also involved in inhibiting
the DPPH radicals. Compounds such as ascorbic acid, β-carotene, α-carotene, and
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different xanthophylls [7, 38] were detected in Musa sp. and may influence the antioxidant
activity of the extracts.
(a)
(b)
Fig. 3: Total phenolic content of ripe pulp and peel (a) and unripe pulp and peel
(b) at a different microwave power of MAE. Temperature, irradiation time and
solid to liquid ratio were fixed at 70 oC, 60 s and 3:60, respectively.
Figure 3 shows the total phenolic content of ripe and unripe Musa sp., pulp, and peel.
Microwave power of MAE is observed to significantly affect the total phenolic content of
ripe and unripe Musa sp. Moreover, a similar trend is observed for the ripe and unripe
peel, whereby a higher total phenolic content is obtained at 500 W but no further
increments are detected at 800 W, with 268 ± 13.4 mg GA/ 100 g FW and 389 ± 19.5 mg
GA/ 100 g FW, respectively. By contrast, the total phenolic content of the pulp is found to
be the highest at 800 W microwave power, for ripe and unripe (Figure 3). The
dissimilarity of the trend might be the result of the complexity of the pulp matrix structure,
where in MAE, the microwave power that contains irradiation energy enhances the
penetration of the solvent into the pulp matrix. The electromagnetic field offers a rapid
transfer of energy to the solvent and solid matrix through molecular interaction, thus
allowing the components to be extracted [39]. Unlike the peel, the total phenolic content
can be extracted at low power (500 W), which is believed to be the result of the less
complex structure of the peel.
Phenolic compounds are essential to fruit constituents because they exhibit
antioxidant activity by inactivating lipid free radicals or preventing decomposition of
hydroperoxides into free radicals [40]. The findings show that the total phenolic content in
the extracts was inversely correlated with the antioxidant activity, especially for the unripe
Musa sp. (Fig. 3b and Fig. 2b), which further shows that the MAE conditions that obtain
high antioxidant activity are not selective for phenolic content.
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3.3 Effect of Irradiation Time on Antioxidant Activity and Total Phenolic Content
The yield of antioxidant activity and the total phenolic content were investigated on
the pulp and peel of ripe and unripe Musa sp. Figures 4 and 5 demonstrate the antioxidant
activity and the total phenolic content of pulp and peel of ripe and unripe Musa sp.,
respectively.
(a)
(b)
Fig. 4: Antioxidant activity of ripe pulp and peel (a) and unripe pulp and peel
(b) at different extraction time of MAE. Temperature, microwave power
and solid to liquid ratio were fixed at 70 oC, 800 W and 3:60, respectively.
In general, the longer the extraction time; hence, the greater the recovery of the
antioxidant activity. The ripe pulp is represented by the trend shown in Fig. 4a. The
antioxidant activity was high at a longer extraction time (90 s), where the inhibition of free
radical was observed to increase from 32% to 88%, at 30 s to 90 s extraction time.
However, prolonging the extraction time did not improve the antioxidant activity for an
unripe peel (Fig. 4b). This phenomenon might be the result of the breakdown of
antioxidant compounds in the less complex matrix structure of unripe peel. Longer
extraction time coupled with higher microwave power might increase the temperature of
the solvent, thus causing the cell wall of the peel to be ruptured [20]. In addition, longer
exposure of the sample to the solvent and microwave irradiation may stimulate extraction
of other components (such as polysaccharides and proteins) of the Musa sp., which then
relatively reduced the antioxidant activity in extracts [41-42]. As a result, extending the
irradiation time with a higher microwave power may lead to thermal degradation of the
phenols [29].
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(a)
(b)
Fig. 5: Total phenolic content of ripe pulp and peel (a) and unripe pulp and peel
(b) at different extraction time of MAE. Temperature, microwave power
and solid to liquid ratio were fixed at 70 oC, 800 W and 3:60, respectively.
Figure 5 shows the total phenolic content of ripe and unripe Musa sp., pulp and peel at
different extraction times. The figure reveals similar trend for ripe and unripe Musa sp.,
where the total phenolic content increased with the increase of the extraction time. From
the figure, phenolic content was found to be significantly higher in unripe Musa sp. in
contrast to ripe Musa sp., where the total phenolic contents were 477 ± 23.9 mg GA/ 100 g
FW and 247 ± 12.4 mg GA/ 100 g FW, respectively. This finding is in agreement with
Fatemah et al. (2012), where the total phenolic content was generally higher in the unripe
than in ripe, whereas the peel contains higher total phenolic content than the pulp [34].
The variation in total phenolic content among different plant materials might be indicated
by several factors such as natural chemical composition, maturity at harvest, soil state, and
conditions of post-harvest storage [43].
3.4 Effect of Solid to Liquid Ratio on Antioxidant Activity and Total Phenolic
Content
Another factor that significantly affected the amount of the antioxidant and total
phenols is solid to liquid ratio. The effect of solid to liquid ratio in MAE was identified
based on the yield of antioxidant activity as well as the total phenolic content of pulp and
peel of ripe and unripe Musa sp. Figures 6 and 7 show the antioxidant activity and the total
phenolic content of pulp and peel of ripe and unripe Musa sp., respectively.
The solid to liquid ratio of 2:60 of ripe Musa sp. resulted in higher antioxidant activity
with the free radical inhibition of 82% when compared to the other ratios (ratio 1:60 and
3:60) (Fig. 6a). Hence, this further explains that the volume of solvent extraction was
enough to swell the ripe Musa at the solid to liquid ratio of 2:60, which caused the cell of
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ripe Musa to directly absorb the microwave energy. As a result, the cell was ruptured, and
the antioxidant was consequently released to the surrounding [44]. In contrast, the
antioxidant activity was increased with the increase of solid to liquid ratio in the unripe
Musa sp. (Fig. 6b).
(a)
(b)
Fig. 6: Antioxidant activity of ripe pulp and peel (a) and unripe pulp and peel
(b) at different solid to liquid ratio in MAE. Temperature, irradiation time
and microwave power were fixed at 70 oC, 60 s and 800 W, respectively.
On the other hand, the total phenolic content of ripe and unripe Musa sp. was
significantly increased with the increase of solid to liquid ratio (Fig. 7). The total phenolic
content was increased from 200 ± 10 mg GA/ 100 g FW to 287 ± 14.4 mg GA/ 100 g FW,
at solid to liquid ratio of 1:60 and 3:60 (unripe pulp), respectively. The experimental data
suggested that the presence of an excessive amount of the solvent at the ratio of 1:60
(solid to liquid) may absorb more energy, and the material will reduce the microwave
absorption. Therefore, the outcome leads to less efficient extraction and lower phenolic
content in the Musa sp. extract [45].
Therefore, it is very crucial to find the best solvent mixture ratio in order to get higher
extraction yield. An optimised sample to liquid ratio could pinpoint the importance of
extracting the target compounds or extracts.
4. CONCLUSION
The results of this study indicate that Musa sp. has a great potential to be utilized as a
natural antioxidant and phenolic compound sources. Higher antioxidant and total phenolic
content were obtained from the peel in comparison to the pulp of Musa sp. The results also
suggested that antioxidant and total phenolic content of Musa sp. extracted using MAE
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(a)
(b)
Fig. 7: Total phenolic content of ripe pulp and peel (a) and unripe pulp and peel
(b) at different solid to liquid ratio in MAE. Temperature, irradiation time
and microwave power were fixed at 70 oC, 60 s and 800 W, respectively.
with toxicity free solvent (distilled water) set in better extraction compared to the
conventional extraction coupled with methanol as obtained by Shaida et al. [26]. The total
phenolic content in this study (similar species to Shaida et al. [26]) was 87.3 mg GA/ 100
g FW, whereas the total phenolic content with methanol was found as 0.03 mg GA/ 100 g
FW for ripe pulp. Hence, this experiment demonstrated that Musa sp. extracted using
MAE with solvent-free toxicity is a better extraction method for natural antioxidant and
phenolic content. Consequently, temperature, microwave power, irradiation time, and
solid to liquid ratio play a huge role in affecting the efficiency of the MAE.
ACKNOWLEDGEMENT
This work was funded by research grant granted by Universiti Malaysia Pahang research
grant no. RDU 150317.
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