Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying

Abstract

In this study, phytochemical compounds were extracted and encapsulated from medicinal plants such as M. oleifera, S. androgynus, and S. grandiflora using subcritical water and the electrospraying technique. The extraction was conducted at temperatures of 120 to 160 °C at various extraction pressures from 1 to 10 MPa in semi-batch systems with a 1.0 mL min⁻¹ flow rate. Under these conditions, the starting materials, that is the medicinal plants, underwent thermal cleavage, allowing the removal of their components. The Fourier transform infrared spectroscopy (FT-IR) spectra of the solid residues indicated that phytochemical compounds were successfully extracted from these medicinal plants. The results revealed that the amounts of extracted phenolic compounds did not increase linearly with increasing extraction temperatures and pressures. The amounts of extracted phenolic compounds could approach 82.26 (140 °C, 5 MPa), 75.32 (160 °C, 5 MPa), and 78.91 (160 °C, 10 MPa) mg of gallic acid equivalents (GAE)/g of dried samples for M. oleifera, S. androgynus, and S. grandiflora, respectively. When the extracted phytochemical compounds were encapsulated with polyvinylpyrrolidone (PVP) via the electrospraying technique, the particle products seemed to exhibit spherical morphologies with diameters less than 1 μm, and the FT-IR spectra of these particle products showed that the medicinal plant extracts were successfully encapsulated by PVP through this technique.

Full text

Phytochemical compounds extraction from
medicinal plants by subcritical water and its
encapsulation via electrospraying
Siti Machmudah
a,*
, Meika Wahyu Fitriana
a
, Nadhia Fatbamayani
a
,
Wahyudiono
b
, Hideki Kanda
b
, Sugeng Winardi
a
, Motonobu Goto
b
a
Department of Chemical Engineering, Institut Teknologi Sepuluh Nopember, Kampus ITS Sukolilo, Surabaya 60111, Indonesia
b
Department of Materials Process Engineering, Nagoya University, Furo–cho, Chikusa–ku, Nagoya 464-8603, Japan
Received 15 May 2021; revised 24 June 2021; accepted 18 July 2021
Available online 05 August 2021
KEYWORDS
Extraction;
Encapsulation;
Electrospraying;
Phytochemical;
Medicinal plant
Abstract In this study, phytochemical compounds were extracted and encapsulated from medici-
nal plants such as M. oleifera,S. androgynus, and S. grandiflora using subcritical water and the elec-
trospraying technique. The extraction was conducted at temperatures of 120 to 160 °C at various
extraction pressures from 1 to 10 MPa in semi-batch systems with a 1.0 mL min
1
flow rate. Under
these conditions, the starting materials, that is the medicinal plants, underwent thermal cleavage,
allowing the removal of their components. The Fourier transform infrared spectroscopy (FT-IR)
spectra of the solid residues indicated that phytochemical compounds were successfully extracted
from these medicinal plants. The results revealed that the amounts of extracted phenolic com-
pounds did not increase linearly with increasing extraction temperatures and pressures. The
amounts of extracted phenolic compounds could approach 82.26 (140 °C, 5 MPa), 75.32 (160 °C,
5 MPa), and 78.91 (160 °C, 10 MPa) mg of gallic acid equivalents (GAE)/g of dried samples for
M. oleifera, S. androgynus, and S. grandiflora, respectively. When the extracted phytochemical com-
pounds were encapsulated with polyvinylpyrrolidone (PVP) via the electrospraying technique, the
particle products seemed to exhibit spherical morphologies with diameters less than 1 lm, and
the FT-IR spectra of these particle products showed that the medicinal plant extracts were success-
fully encapsulated by PVP through this technique.
Ó2021 THE AUTHORS. Published by Elsevier BV on behalf of Faculty of Engineering, Alexandria
University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
1. Introduction
Water is called the universal solvent because it dissolves a lar-
ger number of substances than any other chemical solvent
does. Water possesses high polarity and a high dielectric con-
stant under ambient conditions. Consequently, water was con-
*Corresponding author.
E-mail address: machmudah@chem-eng.its.ac.id (S. Machmudah).
Peer review under responsibility of Faculty of Engineering, Alexandria
University.
Alexandria Engineering Journal (2022) 61, 2116–2128
HOSTED BY
Alexandria University
Alexandria Engineering Journal
www.elsevier.com/locate/aej
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https://doi.org/10.1016/j.aej.2021.07.033
1110-0168 Ó2021 THE AUTHORS. Published by Elsevier BV on behalf of Faculty of Engineering, Alexandria University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

sidered to be a suboptimal extraction solvent for organic or
non-polar compounds under ambient conditions, because it
did not readily dissolve the majority of the phytochemical
compounds derived from plant biomass. The chemical and
physical properties of water change drastically when its tem-
perature is maintained above its boiling point (100 °C) and
below its critical point (374 °C), and the pressure is regulated
to maintain water in the liquid phase. Water in this state is
called subcritical water [1–3]. At 250 °C, the dielectric constant
of water is approximately 27, which is similar to those of etha-
nol (e= 24) and methanol (e= 33) at 25 °C. Therefore, water
in this state may act similarly to ethanol or methanol and
extract a wide range of phytochemical compounds from plant
biomass.
Moringa oleifera (M. oleifera) is a Moringaceae family
plant, commonly known by the names Moringa, horseradish
plant, drumstick plant, benzoil plant, or ben oil plant. All parts
of this plant (leaf, gum, seeds, oil, flowers, roots, and bark) are
edible, and humans have consumed this plant for a long time.
M. oleifera is native to northwestern India, and is widely dis-
tributed and cultivated in subtropical and tropical zones
[4,5]. Moringa has been used worldwide as a traditional med-
icine for diverse health conditions, including abnormal blood
pressure, headaches, swelling, glandular fever, infections, cho-
lera, chest congestion, catarrh, bronchitis, blood impurities,
blackheads, asthma, anxiety, anemia, skin infections, preg-
nancy, diabetes, lactation, intestinal worms, tuberculosis,
sprain, sore throat, semen deficiency, scurvy, respiratory disor-
ders, psoriasis, pimples, pain in joints, and hysteria [4–7].
In this work, subcritical water treatment was applied to
extract phytochemical compounds from M. oleifera leaves
via liquefaction in a semi-batch process. The liquefaction of
plant biomass such as M. oleifera leaves using subcritical water
generally occurs through two processes: (1) the dissolution of
plant biomass in water, and (2) the transformation of the ini-
tial liquefaction products into light products [1,8,9]. When
M. oleifera leaves were subjected to subcritical water treat-
ment, thermal cleavage of M. oleifera leaves occurred, result-
ing in the formation of various radicals. This process is
affected by the liquefaction conditions (the absence or presence
of a donor solvent) because these radicals might be stabilized
as liquefaction products by hydrogen transferred from a donor
solvent. Conversely, no hydrolytic yield of plant biomass mate-
rials is obtained at temperatures lower than 100 °C[8–11].
Kodama et al. [8,9] and Matsunaga et al. [10,11] demonstrated
that subcritical water is an effective solvent for the extraction
of hemicelluloses containing b-glucan compounds from G.
lucidum and barley grain, respectively, via autohydrolysis in
semi-batch processes. Matshediso et al. [12] also employed
pressurized hot water to extract essential substances from pow-
dered dried leaves of M. oleifera. They reported that pressur-
ized hot water can be employed to isolate the essential
substances from nutrient-dense M. oleifera leaves, to improve
the food value. Recently, Nuapia et al. [13] demonstrated the
efficacy of subcritical water by employing pressurized hot
water to recover macro- and micro-nutrients from powdered
dried leaves of M. oleifera. In a subcritical water extraction
system operating in a semi-batch process, the most effective
method of preventing the degradation of the extracted com-
pounds is tuning the residence time (the duration of contact
between liquid water, which is used as the extractant, and
the biomass that constitutes the feed) [14–16].
However, in addition to the instability exhibited by some
phytochemical compounds during their extraction from bio-
mass using subcritical water, the conditions required for their
storage present major hindrances to their end-use. To over-
come these hindrances, the extraction products have been
encapsulated in polyvinylpyrrolidone (PVP) by electrospraying
[17–21]. In addition to being versatile and simple, this encapsu-
lation technique does not require a high-temperature environ-
ment. Additionally, because the collected particulate products
are immediately dried during the electrospraying process, fur-
ther drying is unnecessary. Size-tunable synthesis of monodis-
perse particles in the nanometer to micrometer particle size
range can be performed. During the electrospraying process,
the charges that are generated and subsequently accumulated
on the droplet surface do not affect the properties of the poly-
mer and the phytochemical compound [17,22]. In this work,
the extraction and encapsulation of phytochemical compounds
by subcritical water treatment and electrospraying techniques,
respectively, have also been applied to S. androgynus and S.
grandiflora, which also possess pharmacological properties
and are rich in phytochemical compounds [2,22–27].S. androg-
ynus is a shrubby plant that is widely distributed in South and
Southeast Asia, where it is named daun katuk, phak wan ban,
cekur manis, binahian, dom nghob, or sweet leaf. This shrubby
crop belongs to the Euphorbiaceae family and can grow at
high temperatures and under humid conditions. It contains
high concentrations of bioactive compounds such as phenols,
flavonoids, alkaloids, tannins, fatty acids, steroids, volatile
oils, and terpenoids. In addition to its high food value, S.
androgynus can be employed as a lactation aid, and to treat
gastrointestinal diseases, eye diseases, diabetes, and obesity
[22,27].S. grandiflora is also recognized as an edible medicinal
crop that is widely distributed in Asian countries. S. grandi-
flora leaves can be used for medicinal purposes because they
contain tannins, terpenoids, steroids, glycosides, and alkaloids.
Compounds such as dioctyl ester, 9-hexadecenol, palmitic
acid, 4-methyloxazole, vitamin E acetate, 2-
furancarboxaldehyde, erucic acid, and 3,4,5-
Trimethoxyphenol, were found to be the principal constituents
of S. grandiflora leaves using gas chromatography–mass spec-
trometry. Therefore, S. grandiflora can be used for its anti-
inflammatory, analgesic, anticancer, hypolipidemic,
antiurolithiatic, antidiabetic, and cardioprotective properties
[24–26]. The size-tunable encapsulation technique developed
in this work can provide adequate protection to sensitive phy-
tochemical compounds against decomposition and oxidation
during extraction.
2. Materials and methods
2.1. Materials
M. oleifera,S. androgynus, and S. grandiflora were purchased
from a local market in Surabaya, Indonesia. They were washed
using tap water and dried using a freeze-dryer (Eyela FDU-
1200, Rikakikai Co. Ltd., Tokyo, Japan). Thereafter, these
dried medicinal plants were finely ground using a pulverizer.
The powders obtained from pulverization were sifted through
a 0.65 mm sieve and stored at room temperature in a vacuum
desiccator until they were used in the subsequent step of the
experiments. Polyvinyl pyrrolidone (PVP, (C
6
H
9
NO)
n
; MW:
Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying 2117

29000) was purchased from Sigma-Aldrich Co. (St. Louis,
MO, USA) and used as an encapsulant without further modi-
fication. It was dissolved in ethanol at a concentration of 6 wt
%. Sodium carbonate (Na
2
CO
3
, 99.8 %), sodium nitrite
(NaNO
2
, 98.5%), aluminum chloride hexahydrate (AlCl
3
6H
2
-
O, 98.0%), sodium hydroxide (NaOH, 97.0%), and methanol
(CH
3
OH, 99.7%) were purchased from Wako Pure Chemical
Industries Inc. (Tokyo, Japan). Catechin (C
15
H
14
O
6
,
C217500) was purchased from Toronto Research Chemicals,
Inc. (Toronto, Canada). Folin–Ciocalteu’s phenol reagent,
1,1–diphenyl–2–picrylhydrazyl (DPPH), and gallic acid
(C
7
H
6
O
5
, 97.9 %) were purchased from Sigma-Aldrich (St.
Louis, MO, USA).
2.2. Experimental setup and procedure
Fig. 1 shows a schematic illustration of phytochemical extrac-
tion from medicinal plants using subcritical water in a semi-
batch process. The main components of this extraction system
were a high-pressure pump (200 LC Pump, PerkinElmer, Ger-
many) that served as a solvent (deionized water)-delivery sys-
tem, an oven (Linn High Therm GmbH, model VMK 1600,
Germany) that increased the operating temperature; an extrac-
tor (10 mL in volume; Thar Design Inc., USA) in which the
starting materials were placed, and back-pressure regulators
(BPR; AKICO, Japan) to regulate the operating pressure.
The extraction experiment was performed as follows: Initially,
an extractor containing 4.6 g of a starting material was placed
in the oven. Thereafter, deionized water was delivered via a
high-pressure pump to the extractor at room temperature for
a few seconds to replace the air. Subsequently, it was pressur-
ized to 1–10 MPa. The oven was activated to increase the oper-
ating temperature when the extraction system reached the
desired pressure. In this extraction system, a coil preheater
made from a 1/8 in. stainless-steel tube (SUS316, 50 mL) was
employed to heat the deionized water prior to its entry into
the extractor via a 1/16 in. stainless-steel tube connector. The
preheater was located inside the same oven.
The experiments were carried out at 120–160 °C, during
which, the temperatures of the oven interior, extractor, and
preheater were monitored and measured using a temperature
controller (OMRON E5CJ, Japan) and a K-type thermocou-
ple, respectively. The flow rate of deionized water was set at
1.0 mL min
1
. Each experiment was conducted for 180 min,
and the products of dissolution were collected every 30 min
and rapidly quenched using a double-tube heat exchanger.
During the experiment, light exposure was waived, and each
experiment was carried out in duplicate or triplicate. The solid
residue was dried at 60 °C for 1 d and stored inside a desiccator
at room temperature. The products of dissolution were imme-
diately prepared for the electrospraying process and stored
inside a refrigerator until subsequent analysis. Fig. 2 illustrates
the electrospraying apparatus used to encapsulate the phyto-
chemical compounds using PVP. The apparatus consisted of
a high-voltage power supply (model HGR30-20 N, Japan)
connected to a collector covered with aluminum foil and a
stainless-steel needle of 0.5 mm internal diameter that were
placed inside an enclosed acrylic chamber. The feed solution
was delivered at 0.05 mL/h by a syringe pump (KD Scientific
IC3100, USA) through a plastic disposable syringe. A poly-
ether ether ketone (PEEK) tube of 0.5 mm internal diameter
was used to transfer the feed solution from the outlet of the
plastic disposable syringe to the stainless-steel needle. The dis-
tance between the stainless-steel needle and the electrospun
collector was 10 cm, and the power supply developed a poten-
tial difference of 16 kV between the needle and the collector to
generate an electrostatic force. Each electrospraying experi-
ment was performed at room temperature for 15 min, and
repeated 1–3 times. Prior to the electrospraying process, a mix-
ture of 3 mL of the water-soluble extract and 3 mL of PVP
solution was stirred until complete dissolution. The applied
voltage, the distance between the tip of the stainless-steel nee-
dle and the electrospun collector, and the volume ratio
between the water-soluble extract and the PVP solution were
constant throughout the course of this work. The yields of
the electrospun products were not determined. The as-
obtained electrospun particles were immediately stored inside
a vacuum desiccator at room temperature until subsequent
analysis.
2.3. Analytical methods
The content of phytochemical compounds in the liquid prod-
ucts was analyzed using a Genesys 10 UV–Vis Scanning Spec-
trophotometer (Thermo Fisher Scientific, Waltham, USA).
The concentrations of carbohydrates, proteins, fats, and ash
were not determined. At each extraction condition, the solid
residue was collected and characterized by Fourier-transform
infrared (FT-IR) spectroscopy using a Spectrum Two FT-IR
spectrophotometer (PerkinElmer Ltd., England) to ascertain
Fig. 1 Subcritical water extraction experimental apparatus scheme.
2118 S. Machmudah et al.

the structure of the residue following subcritical water treat-
ment. The FT-IR spectrometer was comprised of a standard
optical system with potassium bromide (KBr) windows and a
universal attenuated total reflectance (UATR) sampling acces-
sory for spectral data collection. The solid residues were placed
directly between the two KBr window disks. The scanning
wavenumber ranged from 4000 to 400 cm
1
, with a step size
of 4 cm
1
. This device was also used to characterize the func-
tional groups present in the electrospun products obtained
from the electrospraying process. Because PVP and the
extracted bioactive compounds were completely soluble in
water, the physicochemical properties of the electrospun prod-
ucts, such as water-solubility and rate of dissolution, were not
verified. The electrospun products were gold-coated using an
IB-3 TEM/SEM specimen preparation apparatus (Eiko Engi-
neering, Japan), and subsequently, their morphologies were
observed by scanning electron microscopy (SEM) using a
JSM–6390LV scanning electron microscope (JEOL, Japan).
2.4. Determination of total phenolic content
The total phenolic content of each extract was determined col-
orimetrically using Folin–Ciocalteu’s reagent. Prior to the
addition of sodium carbonate solution (7.5% w/v) and
Folin–Ciocalteu’s reagent, a 0.1 mL aliquot of the extract
was diluted using 2 mL deionized water, and the concentration
of the resulting solution was measured using a UV–Vis spec-
trophotometer. Thereafter, 1 mL of the aforementioned
diluted solution was transferred into a specimen vial and com-
pletely mixed with 5 mL of Folin–Ciocalteu’s reagent. Prior to
this operation, Folin–Ciocalteu reagent was mixed with deion-
ized water at a ratio of 1:10, and shaken for 3 min using a
Vortex-Genie 2SI-0286 vortex mixer (Scientific Industries,
Inc., USA). Thereafter, 4.0 mL sodium carbonate solution
was added to the solution and completely mixed. The resulting
solution was placed in darkness for 2 h prior to measuring its
absorbance at 765 nm using a single-beam UV–Vis spec-
trophotometer [28,29]. Pure methanol was used as the blank
solution to perform the initial calibration. The absorbance val-
ues of the extracts were compared to a standard calibration
curve produced from the absorbance values of gallic acid of
several known concentrations ranging from 0 to 200 ppm, to
ascertain its concentration in milligrams of gallic acid equiva-
lents (GAE) per milliliter (see Fig. 3). The total phenolic con-
tent was converted into milligrams of GAE per gram of dried
sample, and the measurement for each sample was carried out
in triplicate.
2.5. Determination of total flavonoid content
The total flavonoid content in each aqueous extract was deter-
mined by a colorimetric assay and expressed as milligrams of
catechin equivalents (CE) per gram of dried sample [28,29].
The standard calibration curve was constructed from catechin
solution of multiple known concentrations (see Fig. 4). Ini-
tially, 250 lL of the extract was mixed with 2.5 mL deionized
water. This was followed by the addition of approximately
75 lL NaNO
2
solution (5%, w/v) to the aforementioned solu-
tion. The resulting solution was homogenized using a vortex
mixer for 3 min, and stored under ambient conditions in dark-
ness for 6 min. Thereafter, 0.15 mL AlCl
3
6H
2
O solution
(10%, w/v) was added and homogenized using a vortex mixer
for 3 min under ambient conditions in darkness. The homoge-
nized mixture was stored under ambient conditions in darkness
for 5 min. Thereafter, 0.5 mL NaOH solution (1 M) was added
to it, followed by the addition of deionized water until the total
Fig. 2 Schematic of the electrospraying apparatus (1. Syringe, 2. Syringe pump, 3. High voltage power source, 4. Acrylic chamber, 5.
Needle, 6. Polymer particles, 7. Collector).
Fig. 3 Calibration curve for gallic acid concentration vs.
absorbance.
Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying 2119

volume of the mixture was 3.5 mL. The absorbance of the mix-
ture at 510 nm was measured in triplicate by a single-beam
UV–vis spectrophotometer using an aqueous extract as the
blank solution.
2.6. Determination of antioxidant efficiency
The antioxidant efficiency of the extracts was determined
based on their scavenging effect on the stable DPPH free-
radical activity. A 25 ppm DPPH solution was prepared and
stored in darkness at 4 °C inside a refrigerator. Two milliliters
of extract samples obtained at different concentrations were
added to 800 lL of a methanolic solution of DPPH and sha-
ken vigorously using a vortex mixer. Following 30 min of incu-
bation in darkness at room temperature, the absorbance at
516 nm was averaged from three measurements taken by a
single-beam UV–Vis spectrophotometer using methanol as
the baseline. A blank solution (control) containing the same
amount of methanol and DPPH was also prepared. The per-
centage of DPPH remaining was calculated using equation
(1) [30].
%DPPH remaining = [(AS/AB) 100] ð1Þ
where AB is the absorbance of the control, and AS is the
absorbance of the extract sample. The percentage of DPPH
remaining is negatively proportional to the antioxidant con-
centrations, and the concentration at which 50% of the DPPH
was inactivated was defined as EC
50
. The percentage of DPPH
remaining was plotted against time, and the time required to
achieve steady state at EC
50
was defined as T
EC50
. The antiox-
idant efficiency (AE) was calculated using Equation (2) [31,32].
AE = [1/(EC50 TEC50)] ð2Þ
2.7. Statistical analysis
Each extraction experiment was carried out in duplicate or
triplicate, and the results are presented as the mean ± stan-
dard deviation. Linear regression analysis was used to deter-
mine the total phenolic and flavonoid contents in the extract.
The variance of the experimental results was evaluated using
Microsoft Excel’s ANOVA, with the lower limit for
statistically-significant differences set as 5% (p0.05).
3. Results and discussion
As mentioned earlier, when medicinal plants are treated with
subcritical water, their components may decompose, yielding
Fig. 5 FT-IR spectra of medicinal plants before and after
subcritical water treatment.
Fig. 4 Calibration curve for catechin concentration vs.
absorbance.
2120 S. Machmudah et al.

their backbone units. This treatment may also result in the for-
mation of numerous radicals via thermal cleavage. The carbon
bonds (C–O and C–C bonds) found in esters and ethers might
be broken upon exposure to subcritical water. The same phe-
nomenon might occur for aliphatic C–H bonds. Therefore,
the chemical structures of medicinal plants may be trans-
formed following subcritical water treatment. In this study,
the chemical structures of medicinal plants following subcriti-
cal water treatment were characterized using FT-IR spec-
troscopy. Fig. 5 shows the FT-IR spectra of medicinal plants
before and after subcritical water treatment at 1 MPa at vari-
ous extraction temperatures. Each spectrum originated from
the spectral average of five or more scans. Table 1 summarizes
the infrared signals of the typical functional groups with the
compounds that are generally found in plant biomass
[8,9,11]. As illustrated in Fig. 5, the spectral characteristics of
these medicinal plants are nearly identical, which indicates that
the extraction of phytochemical compounds from these plant
biomasses by subcritical water does not shift the distribution
of their functional groups. However, as shown in this figure,
the peak intensities in the spectra of the medicinal plants were
altered by the treatment. At each extraction condition, the
starting material, consisting of the medicinal plant, exhibited
higher peak intensities compared to its solid residue. The
declining peak intensities in the FT-IR spectra indicated that
this technique successfully extracted these medicinal plant
components.
The band in the 3292.65–3284.94 cm
1
region, which was
associated with OH stretching (intermolecular hydrogen bond-
ing), appeared in every spectrum (M. oleifera,S. androgynus,
and S. grandiflora, and their residues), and its intensity
decreased following subcritical water treatment, particularly
at 160 °C, due to the consumption of alcoholic groups through
the dehydration reaction during extraction at these tempera-
tures [8–11]. Identical trends were evident for the following
bands: 2917.76–2848.76, 1622.10–1618.46, 1435.07–1416.65,
1243.34–1239.50, and 1031.68–1027.31 cm
1
, which were asso-
ciated with the stretching of aliphatic and aromatic C–H
groups, the stretching of aromatic C = C groups, the bending
of acid O–H bonds, the stretching of aryl–alkyl ether linkage
C–O–C groups, and the stretching and deformation of C–O
groups, respectively. The functional groups in these bands
were also expelled and consumed further with increasing
extraction temperature.
Fig. 6 shows the extraction yields from medicinal plants cal-
culated at the end of each experiment when the extraction pro-
cess was performed at temperatures of 120–160 °C and
pressures of 1–10 MPa. Subcritical water treatment is the most
commonly applied method for isolating lignocellulosic bio-
mass components by the cleavage of intra- and inter-
molecular units in or between cellulose, lignin, and hemicellu-
lose via autohydrolysis [8–11]. Therefore, the cellular structure
of lignocellulosic biomass, including that of medicinal plants,
might be disrupted, expelling their components, which permit-
ted dissolution in subcritical water. Consequently, as shown in
Fig. 6, the solubilization of the disruption products occurred at
these extraction temperatures, and the extraction yield
appeared to increase significantly at constant pressure as the
extraction temperature increased from 120 to 160 °C. Statisti-
cal data analysis using two-factor ANOVA showed that both
temperature and pressure significantly influenced the extrac-
tion yield (pless than 0.05) for each of the medicinal plants.
These results also indicate that the extraction temperature,
which is the main variable of the subcritical water extraction
process, may accelerate the extraction process to transform
water-insoluble medicinal plant substances into water-soluble
products through liquefaction [2,8–11,33,34]. Giombelli et al.
[34] reported that increasing the extraction temperature may
enhance the efficiency of extraction using subcritical water,
when they conducted experiments on peach palm by-product
valorization in a semi-continuous process at temperatures of
100–130 °C. They reported that the diffusivity of liquid water
in the matrix increases with increasing extraction temperature,
which can enhance the solubility of the solute in liquid water.
Bordoloi and Goosen [33] also reported that a higher extrac-
tion temperature significantly enhances the extraction yield
and may have a dominating influence on the same when they
employed subcritical water to extract bioactive compounds
from Ecklonia maxima matrix at temperatures of 100–
180 °C. In contrast, although the extraction pressure may pro-
mote the disruption of the cell wall of medicinal plants by liq-
uid water, which can enhance the mass transfer of the
medicinal components to the liquid water solvent, it appears
that increasing the extraction pressure did not exert a strong
influence the extraction yield. This observation is in agreement
with the results of previous works [1,2,8–11,33,34], wherein
increasing the extraction pressure of the subcritical water sys-
tem was ignored because it did not change the physical form of
the water. In other words, the sole function of the extraction
pressure in the subcritical water extraction process is to main-
tain water (the solvent) in its liquid form.
Phenolic compounds are among the most important sec-
ondary metabolites owing to their active roles in reproduction,
physiological processes, and morphological development.
These compounds can be found in plant biomass, including
medicinal plants, and they are formed via the phenyl-
propanoid, shikimate, and pentose phosphate synthetic path-
ways [35]. Phenolic compounds consist of at least one phenol
ring in which the hydrogen atom is generally replaced by an
acetyl, methyl, or hydroxyl unit. Therefore, this phytochemical
Table 1 The FT-IR spectral peak assignment of plant
biomass.
Wave number
[cm
1
]
Band assignment
3600–3000 O–H stretching (Acid, methanol)
2860–2970 C–H
n
stretching (Alkyl, aliphatic, aromatic)
1700–1730, 1510–
1560
C = O stretching (Ketone and carbonyl)
1632 C = C (Benzene stretching ring)
1613, 1450 C = C stretching (Aromatic skeletal mode)
1470–1430 O–CH
3
(Methoxyl–O–CH
3
)
1440–1400 O–H bending (Acid)
1402 C–H bending
1232 C–O–C stretching (Aryl-alkyl ether linkage)
1215 C–O stretching (Phenol)
1170, 1082 C–O–C stretching vibration (Pyranose ring
skeletal)
1108 O–H association (C–OH)
1060 C–O stretching and C–O deformation (C–OH
(ethanol))
700–900 C–H (Aromatic hydrogen)
700–650 C–C stretching
Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying 2121

compound can be generated through autohydrolysis from
plant biomass components using subcritical water [34,36].Xu
et al. [36] reported that phenolic compounds can be formed
by the decomposition of lignin (by breaking the lignin aryl
ether linkages) using subcritical water. They also reported that
the conversion of other biomass components such as carbohy-
drates or proteins using subcritical water can also yield pheno-
lic compounds. Fig. 7 shows the total recovery of phenolic
compounds in the extract obtained from medicinal plants at
temperatures of 120–160 °C and pressures of 1–10 MPa. Gen-
erally, in subcritical water extraction, increasing the extraction
Fig. 7 Recovery of total phenolic content under different
extraction conditions.
Fig. 6 Extraction yields of medicinal plants under different
conditions.
2122 S. Machmudah et al.

temperature enhances the extraction efficiency of phenolic
compounds, while increasing the extraction pressure facilitates
the process of extraction. Higher extraction temperatures pro-
mote the cleavage of phenolic compound bonds in the plant
biomass, and higher extraction pressures increase the contact
between the plant biomass and the subcritical water. As a
result, the amount of water penetrating into the plant biomass
cells increases, which increases the internal pressure of the
cells. Eventually, this process pushes the dissolved substances
out of the cells through the pores in the cell walls [37].
Fig. 8 Recovery of total flavonoid content under different
extraction conditions.
Fig. 9 Antioxidant efficiency under different extraction
conditions.
Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying 2123

As shown in Fig. 7, the total phenolic content recovery did
not increase linearly with increasing extraction temperature
and pressure. The quantity of total phenolic compounds was
approximately 42.26 mg GAE/g dried S. grandiflora sample
when the extraction process was performed at a temperature
of 140 °C and a pressure of 5 MPa. Under identical extraction
conditions, the quantity of total phenolic compounds
approached 82.26 mg GAE/g dried sample when M. oleifera
was used as the starting material. Based on these results, we
believe that this extraction technique is feasible and capable
of extracting phenolic compounds from M. oleifera,S. androg-
ynus, and S. grandiflora.
Flavonoids are also important natural products that are
ubiquitous in plant biomasses, and they are also recognized
as secondary plant metabolites that consist of an aromatic ring
containing at least one hydroxyl unit [38]. As mentioned ear-
lier, water at subcritical conditions has been employed to
extract phytochemical compounds including flavonoids from
medicinal plants through liquefaction in semi-batch processes.
There are several steps to extract this phytochemical from
these medicinal plants using this technique [1,8,39]. Initially,
the liquid water contacts and wets the matrix, followed by
the desorption of solutes from the matrix. In this process,
cleavages of chemical bonds or active sites may occur. There-
after, the solutes dissolve in the liquid water and diffuse out of
the matrix. Finally, the solutes will dissolve in the liquid water
acting as an extraction medium. The efficiency of this process
is affected mainly by temperature and pressure, wherein the
temperature may shift the physical/chemical properties of
water in the liquid state, which is maintained by the pressure.
Ko et al. [40] conducted subcritical extraction to recover flavo-
noids from various plant biomasses, such as grapefruit peels,
orange peels, lemons peels, carrots, parsleys, seabuckthorn
leaves, Saururus chinensis, and onion skins, at temperatures
of 110–200 °C. They reported that a higher extraction temper-
ature may increase the thermal agitation and decrease the
strength of hydrogen bonding, resulting in a higher extraction
efficiency. Kim and Lim [41] studied the kinetics of flavonoid
extraction from citrus unshiu peel using subcritical water
(120–180 °C) in a semi-continuous system. They concluded
that the rate of extraction of flavonoids from citrus unshiu peel
increased proportionally with increase in extraction tempera-
ture. However, as shown in Fig. 8, although the total flavonoid
content in the extracts recovered from M. oleifera,S. androg-
ynus, and S. grandiflora did not increase linearly with increas-
ing extraction temperatures and pressures when the extractions
were performed at temperatures of 120–160 °C and pressures
of 1–10 MPa, this extraction process can extract 1.90 mg of
CE/g of dried sample.
Subcritical water is the most promising extractant for iso-
lating various compounds from plant biomass. In addition,
this extraction technique is environment-friendly. Subcritical
water may also promote antioxidant generation [42].Fig. 9
shows the antioxidant efficiency of the extracts of M. oleifera,
S. androgynus, and S. grandiflora when the extractions were
performed at temperatures of 120–160 °C and pressures of 1–
10 MPa. This showed that increasing the extraction tempera-
ture may improve the antioxidant efficiency of the extract, par-
ticularly at a pressure of 5 MPa. Gilbert-Lopez et al. [42]
reported that antioxidant compounds can be generated during
the subcritical water extraction of plant biomass through dif-
ferent processes: i) hydrolysis reaction—since the extraction
was performed at high temperatures in liquid water (between
100 and 374 °C), hydrolysis reactions may occur and lead to
the generation of new compounds that possess antioxidant
properties; ii) decomposition reaction—the original compound
may decompose into antioxidants in the extract; and iii) uncat-
egorized reactions—the extracted components may interact
Fig. 10 SEM images of (a) original PVP and (b) PVP particles generated by electrospraying.
2124 S. Machmudah et al.

and react with each other to form new substances that possess
antioxidant properties. Hence, increasing the extraction tem-
perature may have a positive or negative influence on the
antioxidant efficiency of the extract. As shown in Fig. 9,
increasing the extraction temperature seems to have a positive
effect on the antioxidant efficiency on the extracts of M. olei-
fera,S. androgynus, and S. grandiflora. In particular, at an
extraction pressure of 5 MPa, the antioxidant efficiency
improved with increasing extraction temperature for M. olifera
and S. androgynus. Based on the results, we surmise that this
range of extraction temperatures (120–160 °C) at a pressure
of 5 MPa can endow liquid water with unique characteristics
which may promote various reaction types.
To provide and maintain easier storage of the water-soluble
extracts, they were encapsulated in PVP by electrospraying.
The term ‘‘encapsulation”is used to refer to the process of
encasing water-soluble extracts using PVP, thereby resulting
in particles having diameters in the nano to micro scales.
Encapsulation is a safe and efficient technique to entrap
water-soluble bioactive compounds, and may improve their
delivery to target sites. In this technique, the core, which con-
tains water-soluble bioactive compounds, is surrounded by a
shell made of PVP that envelops and protects the water-
soluble bioactive compounds from the external environment.
Hence, the degradation of water-soluble bioactive compounds
can be avoided [43–45]. In the electrospraying technique, the
feed solution, containing a mixture of the water-soluble
extracts and the PVP solution, is stretched and broken up
using electrical forces during the atomization process to gener-
ate nano- to micro-scale particles. When a high voltage is
introduced into the feed solution that flows out from the stain-
less steel needle, the electric field generates free charges in the
feed solution and the on the surfaces of its droplets. There-
after, the jet is formed and driven from the stainless-steel nee-
dle to the electrospun collector product. Because the charge
distribution and charge quantity on the surfaces of the droplets
are uneven, the jet instabilities break it up into smaller dro-
plets. Under these conditions, rapid evaporation of the feed
solvents occurs concurrently. This generates nano- to macro-
scale solid particles when the droplets approach and impact
the collector [22].
Figs. 10 and 11 show the SEM images of the original PVP
and the PVP particles obtained by electrospraying without and
with water-soluble extracts, respectively. Clearly, the electro-
Fig. 11 SEM images of water-soluble extract/PVP particles (the liquid extract obtained at 140 °C and 5 MPa).
Phytochemical compounds extraction from medicinal plants by subcritical water and its encapsulation via electrospraying 2125

spun products comprised particles without fibers when the feed
solution consisted solely of PVP (see Fig. 10(b)). The size of
the PVP particles obtained by electrospraying was less than
5lm, much smaller than that of the original PVP. This indi-
cates that the PVP particles are generated by electrospraying
using aqueous ethanol as a solvent. In contrast, nascent fibers
were found in the electrospun products when the feed solution
consisted of a mixture of the PVP solution and the water-
soluble extracts that were fed as the starting material (see
Fig. 11). Solution properties, such as viscosity and the nature
of the solvent, may directly affect the electrospraying process
to generate electrospun products [22]. When a feed solution
having low viscosity and high surface tension is fed as the
starting material, the electrospraying system may work prop-
erly to produce electrospun products comprising solid particles
without string formation. Conversely, increasing the viscosity
and decreasing the surface tension of the feed solution may
promote the stabilization of the jet via chain entanglement,
preventing particle generation and improving the fiber content
of the electrospun products. Consequently, as shown in
Fig. 11, nascent fibers were found in the electrospun products
of each sample, probably because of the changes in the compo-
sition of the feed solution that altered its properties [46,47].
However, it appears that the particle diameter obtained from
the mixtures of the PVP solution and the water-soluble
extracts was smaller than 1 lm, that is, smaller than the parti-
cle diameter from the PVP solution only.
Thereafter, the electrospun products were characterized
using FT-IR to observe the PVP molecule structure after the
electrospraying process with or without the presence of
extracted medicinal compounds. In addition to the type of
chemical bonds in the substance, this technique also permits
the identification of unknown component groups. The PVP
raw material was used to provide a reference. Fig. 12 shows
the FT-IR spectra of the PVP raw material (a), the PVP elec-
trospun product (b), and the electrospun product of PVP con-
taining medicinal plant extracts (c–e), when the extraction was
conducted at a temperature of 140 °C and a pressure of 5 MPa.
Differences between the PVP spectrum (PVP raw material) and
the PVP electrospun products spectra (PVP electrospun prod-
ucts with and without medicinal plant extracts) FT-IRwere
marginal, indicating that the electrospraying process did not
shift the PVP functional groups. However, as illustrated in
Fig. 12, a new infrared band appeared in the 1439.58–
1438.61 cm
1
range when the PVP solution containing medic-
inal plant extracts was used as a starting material to produce
particles. This might be induced by the formation of inter-
molecular hydrogen bonds between the OH bending functional
group of the medicinal plant extract and the CH
2
band of PVP
[18,22]. Therefore, we surmise that the medicinal plant extracts
were successfully encapsulated in PVP by electrospraying.
4. Conclusions
Phytochemical compounds were extracted from M. oleifera,S.
androgynus, and S. grandiflora using subcritical water at tem-
peratures of 120–160 °C and pressures of 1–10 MPa in a
semi-batch system. Under these extraction conditions, the
FT-IR spectra showed that subcritical water extracted the
components of these medicinal plants via liquefaction. The
amounts of extracted phenolic compounds approached 82.26
(140 °C, 5 MPa), 75.32 (160 °C, 5 MPa), and 78.91 (160 °C,
10 MPa) mg of gallic acid equivalents (GAE)/g of dried sample
for M. oleifera, S. androgynus, and S. grandiflora, respectively.
Subsequently, these phytochemical compounds were encapsu-
lated in PVP via an electrospraying technique. The SEM
images indicated that the products were spherical particles
having diameters smaller than 1 lm, and the FT-IR spectra
of these electrospun products indicated that the medicinal
plant extracts were successfully encapsulated in PVP using this
technique. Based on the results, we believe that the proposed
process is an effective technique for extracting and encapsulat-
ing phytochemical compounds from plant biomass matrices.
Declaration of Competing Interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Fig. 12 FT-IR spectra of electrospun products.
2126 S. Machmudah et al.

Acknowledgment
This work was supported by the Directorate of Research and
Community Service, Ministry of Research and Technology/
National Research and Innovation Agency of the Republic
of Indonesia through a research grant (‘‘World Class
Research”) [grant number 128/SP2H/AMD/LT/DRPM/2020,
15/Addendum/ITS/2020, 009/SP2H/LT/DRPM/2021, and
1035/PKS/ITS/2021].
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