Production of submicron particles of the antioxidants of mango leaves/PVP by supercritical antisolvent extraction process

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DOI: 10.1016/j.supflu.2018.09.007
Cite this publication
Abstract
Mango leaves contain bioactive compounds of interest, due to their beneficial activity for health as antioxidant among others. These compounds can easily degrade by light or oxygen, for this reason, the encapsulation of antioxidant natural extract from these leaves is suggested as an effective approach against the degradation of phenolic compounds and to achieve a sustained release of them. The antioxidants/PVP encapsulates were obtained by supercritical antisolvent extraction process (SAE) using pressurized liquid extract (PLE). Using different conditions of pressure (120–180 bar), temperature (35–65 °C) and mass ratio (1/3–1/9), spherical particles were obtained in a submicronic range between 0.11–0.59 μm. Antioxidants/PVP ratio was the most significance variable on the particle size and on the time of release of the mangiferin, quercetin 3-D-galactoside and penta-O-galloyl glucose, what means as greater amount of polymer higher particle size and smaller percentage of release.
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Accepted Manuscript
Title: Production of submicron particles of the antioxidants of
mango leaves/PVP by supercritical antisolvent extraction
process
Authors: M.C. Guam´
an-Balc´
azar, A. Montes, C. Pereyra, E.
Mart´
ınez de la Ossa
PII: S0896-8446(18)30483-2
DOI: https://doi.org/10.1016/j.supflu.2018.09.007
Reference: SUPFLU 4369
To appear in: J. of Supercritical Fluids
Received date: 20-7-2018
Revised date: 12-9-2018
Accepted date: 13-9-2018
Please cite this article as: Guam´
an-Balc´
azar MC, Montes A, Pereyra C, de la Ossa
EM, Production of submicron particles of the antioxidants of mango leaves/PVP by
supercritical antisolvent extraction process, The Journal of Supercritical Fluids (2018),
https://doi.org/10.1016/j.supflu.2018.09.007
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Production of submicron particles of the antioxidants of mango leaves/PVP by
supercritical antisolvent extraction process
M. C. Guamán-Balcázar a,b, A. Montes a,*, C. Pereyra a, E. Martínez de la Ossa a
aDepartment of Chemical Engineering and Food Technology, Faculty of Sciences, University of
Cádiz, International Excellence Agrifood Campus (CeiA3)11510 Puerto Real (Cádiz), Spain.
bDepartamento de Química y Ciencias Exactas, Universidad Técnica Particular de Loja,
San Cayetano Alto sn, AP 1101608, Loja, Ecuador
Corresponding author.
Phone: +34 956016264
E-mail address: antonio.montes@uca.es
Graphical Abstract
Highlights
PILOT PLANT
PG1
PG1
Extract
+
PVP
CO2
tank
Cooler
CO2 pump
Solv ent pump
ABPR
MBPR
CO2
Separator
Vessel
Liquid
solution
PLE+PVP
SEM
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450 500
% Relaese Mangiferina
Minutes
Run 3 (1/9) Run 4 (1/9) Run 9 (1/3)
Run 10 (1/3) Run 11 (1/6) PLE precipitate
RELEASE
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Nano and submicron particles of antioxidant/PVP were generated by SAE
The antioxidant/PVP mass ratio was significant on particle size and release profile
Pressure and temperature were not significant variables in the assayed levels
The release of four antioxidants into simulated fluids against time were analysed by UPLC
In general, the higher amount of polymer the higher particle size and the smaller
percentage of release.
ABSTRACT
Mango leaves contain bioactive compounds of interest, due to their beneficial activity
for health as antioxidant among others. These compounds can easily degrade by light or oxygen,
for this reason, the encapsulation of antioxidant natural extract from these leaves is suggested
as an effective approach against the degradation of phenolic compounds and to achieve a
sustained release of them. The antioxidants/PVP encapsulates were obtained by supercritical
antisolvent extraction process (SAE) using pressurized liquid extract (PLE). Using different
conditions of pressure (120 180 bar), temperature (35 65 º C) and mass ratio (1/3 1/9),
spherical particles were obtained in a submicronic range between 0.11 0.59 µm.
Antioxidants/PVP ratio was the most significance variable on the particle size and on the time of
release of the mangiferin, quercetin 3-D-galactoside and penta-O-galloyl glucose, what means
as greater amount of polymer higher particle size and smaller percentage of release.
Keywords: Supercritical antisolvent extraction; encapsulate; mango leaves antioxidants/PVP
1. Introduction
Mango is the dominant tropical fruit tree variety produced in the worldwide. In 2016,
the world production was 47 millions of tons approximately, where Asia continent is the largest
producer (74.3%) followed by America (12.8%), Africa (12.8%) and Oceania (0.1%). Particularly,
Spain had a production of more than 20000 tons which represents 0.04% of the world
production [1]. Both mango fruits and by-products such as stem, bark, leaves, peels and seeds
present antioxidant [24], anti-inflammatory [5], anti-proliferative [6] and anti-cancer [7]
properties. On the other hand, medicinal and pharmacological uses from mango leaves have
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been described in some research as the potential drug for the treatment of malaria [8],
diphtheria and rheumatism [9], due to the high content in polyphenols like phenolic acids,
xanthones, benzophenone derivative and flavonoids [4,10]. The polyphenolics compounds are
secondary products of the metabolism of plants; they are present mainly in fruits and
vegetables. These bioactive compounds are added to human diet [11] due to antioxidant
properties, it has been associated with the prevention of chronic diseases as certain types of
cancer, degenerative disorders, cardiovascular problem, diabetes and osteoporosis [12].
Nevertheless, the effectiveness of polyphenolics depends on preserving the stability against
heat, oxygen, light and moisture [13], as well as, bioactivity and bioavailability of the active
compounds [14]. On the other hand, the unpleasant taste of some phenolic compounds limits
its application in the food and nutraceutical industry, so using encapsulated polyphenols could
improve the sensory characteristics of the obtained product [14].
The objective of encapsulation is to protect the bioactive compounds from
environmental factors as well as improve the useful life and contribute to a sustained release
of the polyphenols. In literature, various physical and physicochemical encapsulation
techniques for natural products are reported [1417]. The most common physical and
physicochemical methods are spray drying [1820], emulsion [21], extrusion [22], fluid bed
coating [23], electrospinning [24], supercritical fluids [17,25,26], coacervation [27],
emulsification-solvent removal [28] and cooling of emulsions [29], nevertheless, the physical
methods are the most used.
In recent years, the encapsulation by supercritical technology using sc-CO2 (supercritical
CO2) is a suitable technique for natural products encapsulation. This process present several
advantages when is compared with other methods as high selectivity, low organic solvent
consumption and sc-CO2 is non-toxic and non-flammable; it is possible to carry out the process
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at low temperature, an important characteristics when it is worked with sensitive compounds
[17,30]; and it is possible to obtain nano and microparticles with controlled dimensions.
Supercritical antisolvent process (SAS) is a specific technique that has been used to micronize
several kind of compounds [31]; in this process the supercritical antisolvent (sc-CO2) is placed
in contact with the solution (solvent + active compound) in the vessel, the supercritical
antisolvent is dissolved in the liquid phase, decreasing its density and the solvation power of
the organic solvent, while the solvent is evaporated in the supercritical phase, which leads to
supersaturation of the solution and then to precipitation of the active compounds. The solvent
excess is removed by a continuous flow of sc-CO2 [15,32].
Some authors have also used the supercritical antisolvent extraction (SAE) method
where the only difference with regard to SAS process is that the solution of active compound,
used in SAS process, is substituted by an ethanolic extract from natural products to obtain co-
precipitates or encapsulates particles of these extracts, such as Oleoresin from C. Frutesces
[33], astaxanthin from Haematococcus pluvialis [34], polyphenols from Rosemary extract [35],
carotenoids from shrimp residue extract [36], curcumin from turmeric extract [37] and
polyphenols and caffeine from green tea [38]. However, research regarding precipitate of
mango leaves antioxidants by supercritical fluids technology is still limited.
The properties of the obtained encapsulated particles depend mainly on the operational
conditions such as pressure, temperature, CO2/liquid solution flow rate ratios, type of
encapsulation agent or carrier and encapsulation agent /active compounds ratio. The aim of
the research was to obtain micro or nanoparticles of encapsulates containing polyphenols of
mango leaves by SAE process using polyvinylpyrrolidone (PVP) as a carrier. PVP is an inactive
ingredient present in FDA-approved drug products. Some investigations have obtained
successful results using (PVP)/drug to obtain micro and nanoparticles such as PVP/Nimesulide
[39], PVP/corticosteroid [40], PVP/liposoluble vitamins [41], PVP/folic acid [42], PVP/diflunisal
[43], PVP/curcumin [44], PVP/astaxanthin [45] and PVP/cefuroxime acetyl [46].
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2. Materials and methods
2.1. Materials
Polyvinylpyrrolidone (PVP, average molecular weight 10 kg/mol), 2,2-Dyphenil-1-
picrylhydrazyl (DPPH), gallic acid, mangiferin and quercetin (≥98%), penta-O-galloyl-β-D-
glucose hydrate (≥96%) and quercetin 3-D-galactoside and 3,4-dihydroxybenzoic acid (≥97%)
were purchased from Sigma-Aldrich (Steinheim, Germany). Ethanol, acetonitrile and formic
acid (HPLC grade), sodium hydroxide, monobasic potassium phosphate, sodium chloride were
supplied by Panreac (Barcelona, Spain). Hydrochloric acid from Global Chem (Seville, Spain).
CO2 with a maximum purity of 99.8% was obtained from Linde (Spain). Double-distilled Milli-
Q grade water was used.
Mangifera indica L. leaves (Kent variety) was collected in 2017 by Finca Experimental
‘La Mayora’, Superior Centre of Scientific Researches (CSIC) (Málaga, Spain) and dried until
drying loss of 91%. The leaves were crushed an overage particle size of 750 µm.
2.2. Preparation of mango ethanolic extracts and polymer solutions
The polyphenols extraction was obtained by pressurized fluid extraction (PLE) for three
hours in SF100 pilot plant (Thar Technologies) using ethanol (ETOH) as a solvent at 120 bar,
80 ºC and 10 g ethanol/min [47]. The final concentration of extract was 20 mg/mL.
For the precipitation experiments, PVP in different ratios (1/3 1/9 extract/PVP w/w)
was dissolved into the PLE extracts in order that 10 mg/mL of antioxidants-PVP was the
concentration of injected solution in the SAS process. In this way, 25 mL of PLE extract (2 5
mg/mL) and 25 mL (15mg/mL 18 mg/mL) of PVP solution at different concentrations were
mixed.
2.3. Supercritical antisolvent extraction (SAE)
The SAE equipment built by Thar Technologies® (SAS200) is shown in Fig. 1. The
precipitation plant includes two high-pressure pumps (B1 and B2), one for CO2 and another for
the extract/PVP solution, a precipitator vessel (2 L), an automated high precision back-pressure
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regulator (V1), a separator (0.5 L) to separate the solvent (ethanol) and CO2 and finally, a
manual back-pressure regulator (V2). As the precipitator vessel (P1) and separator (S1) are
surrounded by an electrical heating jacket. The system was described in detail in a previous
publication [48].
All of the runs were carried out in a semi continuous mode according to the following
experimental protocol: in the first place, CO2 was pumped into the precipitator vessel and
heaters were switched on in order to achieve the desired conditions of pressure and
temperature inside it. Then an ethanolic extract containing the polymer in the selected ratios
was pumped and sprayed through a nozzle of 100 µm of internal diameter into the precipitator
vessel. The spray of the solution generates the formation of small droplets and a rapid mass
transfer occurs between the liquid and CO2 phases provoking the antisolvent effect so the
supersaturation of the liquid solution and the consequent precipitation of the particles of the
extract as a powder on the internal wall of the vessel and/or on the frit. The CO2 was
continuously flowing during one hour after the precipitation step to eliminate the residues of
solvent.
The effects of three independent variables as pressure (120 180 bar), temperature (35
65 ºC) and mass ratio (1/3 1/9 extract/PVP) on particle size distribution were evaluated
using screening (Design class) Factorial 2^3 design, with three central points. The total design
consisted of 11 runs that were carried out randomly. The solution flow rate (5 g solution/ min),
nozzle diameter size (100 µm), CO2 flow rate (30 g CO2/min) and washing time (60 min) were
constants conditions. The operational conditions used for the SAE are shown in Table 1.
2.4. Mean particle size and particle size distribution
Nova NanoSEMTM 450 scanning electron microscope (SEM) was used to evaluate the
morphology and size of the samples. The obtained sample powder by SAE process was covered
with a coating of 15 nm film of gold using a sputter coater. The SEM images were processed
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using Scion image analysis software (Scion Corporation) to obtain the particle sizes.
Approximately 300 particles were analysed in each experiment. The results of mean particle
size and particle size distribution were calculated using software Statgraphics Centurion XVI.
2.5. UPLC analysis
UPLC analysis was performed with a Waters Acquity Ultra Performance Liquid
Chromatography system (Waters, USA) equipped with a Waters XevoG2 QTof MS (Waters
MS Technologies, Milford, MA, USA). Analyses were carried out on an ACQUITY UPLC® with
a Fotodiode Array Detector (PDA, UPLC LG 500 nm) and processed by MassLynx 4.1 version
software with Target Lynx version 4.1 programmes (Waters Corporation, Milford, MA, USA).
A reverse phase C18 column (ACQUITY UPLC BEH® C18 1.7 µm, 2.1 - 50 mm) from Waters
Corporation (Milford, MA, USA) was used and the temperature was set at 40 ºC.
A gradient elution program was used with two phases: A (0.1% acetic acid in water) and
B (0.1% acetic acid in acetonitrile). The applied gradient was as follows (time, A%): 0 - 0.3
min, 98%; 0.3 - 1.5 min, 98 - 65%; 1.5 - 2 min,65 - 0%; 2 - 2.5 min, 0 - 98%; 2.5 - 3 min, 98%.
The column was subsequently washed with 100% A for 6 seconds. The flow rate was set at 0.8
mL/min. The injection volume was set to 2 µL. Compounds were detected at 220 - 500 nm
according to the retention time and using the calibration curves for the different standards. The
antioxidants quantified compounds were gallic acid, mangiferin, quercetin 3-D-galactoside,
3,4-dihydroxybenzoic acid and penta-O-galloyl glucose. All analyses were carried out in
triplicate and the standard deviation (SD) was calculated in each case.
2.6. In vitro release test
The release of three phenolic compounds as penta-O-galloyl glucose, quercetin 3-D-
galactoside and mangiferin in the encapsulated was conducted at 37°C in simulated gastric
(SGF) and intestinal (SIF) fluids. SGF was prepared by dissolving 2 g of sodium chloride with 1
L of water, while that SIF was prepared by mixing of 6.8 g of monobasic phosphate in water
(1L). The gastric and intestinal solution were adjusted to pH 1.2 ± 0.1 (0.2 N HCl) and pH 6.8 ±
0.1 (0.2 N NaOH) respectively [49].
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10 mg of encapsulated samples were weighted and mixed with 40 mL of simulated fluid.
Aliquots of 3 mL were taken at predefined times and replaced with 3 mL of fresh simulated fluid.
The samples were filter and measured in UPLC equipment.
3. Results and discussion
3.1. SAE precipitation of the antioxidants/PVP
A factorial design (2^3) with eleven runs was completed as a screening method to
elucidate the variables that were likely to influence the size of produced particles from mango
leaves extracts. Three variables as mass ratio (x1), temperature (x2) and pressure (x3) with three
level each respectively as follow: 1/3 to 1/9 extract/PVP, 35 to 65 ºC and 120 to 180 bar were
selected. The experiments that were performed are shown in Table 1. From the central point
experiments (runs 1, 8 and 11) it can be calculated the precision of the method as media deviation
that is 0.034. In this sense the precision of the method is adequate due to this value is quite lower
than the difference of the values of the experiments that were compared in this work to find out
the parameter trends. Only if some runs whose comparison do not have interest to be done as the
cases of runs 2 and 6, 7 and 9 or 7 and 10 the particle size values would be compromised. The
values of the estimated main and interaction effects of factors in decreasing order of importance
are plotter in Fig. 2, where observed that mass ratio (x1) found to be significant (p<0,05) for the
particle size, while the temperature (x2), pressure (x3) and interactions of these variables were
found to have no significance (p > 0.05). Additionally, it is shown that the pressure has the lowest
effect on the particle size compared to the mass ratio and temperature variables. On the other
hand, the graphic of main effects (Fig. 3) obtained from the analysis of variance shown that the
smallest particles were obtained by working with lower mass ratio, lower temperature and
higher pressure. Likewise, when performing the one-way simple analyses of variance for each
variable (x1, x2, x3) with respect to particle size, it was determined that only the mass ratio 1/3-
1/9 have significance (p > 0.05), but not between 1/3-1/6 and 1/6- 1/9, also there is no statistical
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difference between the different pressures (120, 150, 180 bar) and different temperatures (35,
50, 65 ºC) assayed.
3.1.1. Effect of mango antioxidants/polymer on particle size
The extract/PVP mass ratio was the principal effect on the particle size. It is so, when
the mass ratio from 1/9 to 1/3 is decreased, the particle size was reduced, this fact can be seen
by comparing the run 3 (0.59 µm) and 10 (0.13 µm), run 4 (0.33 µm) and 9 (0.11 µm), and run 5
(0.44 µm) and 6 (0.21 µm), which are in similar conditions of pressure and temperature (Fig. 4).
Similar results were reported by Marco et al. [41] in the PVP/NIM (Nimesulide) SAS co-
precipitate, where it was indicated that the ratio was an important factor for the PVP/drug fine
particles obtaining. In their research was found that PVP/NIM particles decreased when 3/1
(2.27 µm) as a mass ratio is used instead of 10/1 (4.03 µm). Other authors also concluded that
increasing the extract to polymer mass ratio, a particle size increasing was produced, concretely
from 0.60 µm (1/5) to 3.81 µm (1/20) in folic acid / PVP [42]; from 0.09 µm (1/5) to 0.20 µm
(1/20) in astaxanthin/ PVP [45] and from 0.25 µm (1/1) to 0.36 µm (1/10) in mangiferin/Cellulose
acetate phthalate (CAP) [49]. In this way, in the supercritical antisolvent process, parameters as
viscosity and density of the feed solution are strongly influenced in the formation of the jet, it is
so, when lower viscosities of the solution compounds/PVP, a smaller particle size should be
precipitated [50].
3.1.2. Effect of temperature and pressure
The effect of operating temperature on particle size was investigated using three
conditions 35, 50 and 65 ºC. According to the literature, some authors who have worked
with PVP as a carrier concluded that a reduction in the temperature led to a decrease in
the particle size [41], although other researchers indicated that the influence of
temperature on particle size did not appear to be relevant [51]. In our research, the
temperature variable did not have significantly (p<0.05) influence on the particle size
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(Fig. 2), but comparing the run 3 (0.59 µm) and 7 (0.12 µm), 4 (0.33 µm) and 5 (0.44
µm), and finally 6 (0.21 µm) and 9 (0.11 µm), it can be observed that there is a small
trend (Fig. 5), the lower the temperature, the smaller particle size. It could be due that the
solubility of the compounds in the ethanol increases and the CO2 power solvent is reduced
with temperature, thus the level of supersaturation is reduced and larger particles are
formed [52].
With respect to the pressure, the antioxidants + PVP solutions were precipitated
in the pressure range of 120-180 bar. These conditions were determined considering the
equilibrium phase diagram of CO2 + ethanol binary system (mixture critical point, 71.8
bar, 40 ºC at 0.98 molar fraction of CO2) [53] and the previous information about the best
conditions to precipitate mango antioxidants [10] and PVP [42,46]. In this sense to avoid
the polymer could influence the phase diagram provoking that the experiment took place
in the two phases region and the process failing, the operating conditions of experiments
were situated far above MCP. In our work, when pressure increases from 120 to 180 bar
(run 2 and 9), the particle size slightly increases from 0.11 to 0.18 µm using 1/3 mass
ratio, whilst that in the run 4 (120 bar, 1/9, 0.33 µm) and 7 (180 bar, 1/9, 0.12 µm), it
produces the opposite effect. Indeed, the effect of pressure on the particle size is not still
well defined. As the pressure increases holding the temperature, the density of CO2 and
its solvent power increase provoking a rapid mixing and mass transfer between CO2 and
the ethanolic solution which leads to a supersaturation of this solution and the subsequent
formation of smaller particles. However, at the same time, if the density increases, the
diffusivity of the organic solvent decreases and the mass transfer is slow down which
results in larger particles [42,4446]. The second effect seems to be more dominant in
PVP precipitation as concluded others authors. In this way, Chhouk et al. observed the
size of PVP/curcumin particles slightly increased (81 to 90 nm) when pressure increased
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from 100 to 150 and had not significant change from 150 to 200 bar [54]. And in the same
line, Prosapio et al. [43-44] reported that the obtained particle size increased by increasing
the operating pressure from 90 to 150 bar when preparing PVP/FA (Folic acid) (from 0.18
to 0.65 µm) , PVP/MEN (Menadione) (0.18 µm to 4.08) and PVP/TOC (-tocopherol)
(from 1.8 to 2.64) by supercritical antisolvent process.
3.1.3. Morphology of particles
A reference point about the possible morphologies of the coprecipitates can be deduced
by morphologies observed of single compounds by SAS process [31]. In this sense, initially, both
PLE extract [55] and the PVP were successfully precipitated alone using the supercritical
antisolvent process at 150 bar and 35 ºC, 30 g of CO2/min, 100 µm of nozzle diameter and 5 g of
solution/min. The experiments were carried out to determine the shape and size of these
particles separately. In the Fig. 7 can be seen the morphology of the PLE precipitated in the form
of slightly agglomerated spheres with mean particle diameter size of 0.05 µm [55], whereas the
PVP precipitates have a spherical shape with particle diameter of 0.57 µm.
Likewise, in Fig. 7, can be observed the effect of including PVP in the solution
extract on the particle morphology. It is interesting, that when mango extract and PVP
were precipitated together, spherical particles of smaller diameter sizes were obtained,
compared to the PVP and extract alone due to that the precipitation mechanism and rates
could be different in co-precipitation experiments compared to precipitation of PVP and
mango extracts alone [46]. In this way, other authors highlighted the ability of PVP, in
coprecipitation process, to force the system to spherical particle precipitation [39-42, 56-
57].
Mainly, the morphology of the particles was affected by the mass ratio. Thus,
in 1/3 w/w slightly agglomerated spheres were obtained, while that using 1/9 w/w the
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particles were spherical as can be seen in Fig. 4, especially in run 2 with respect run 7.
These effects could be due to the insufficient amount of polymer for hardening the droplet
into spherical particles [44]. Additionally, in the experiments that were carried out with
1/9 mass ratio, can be observed that the morphology did not change, but the particle size
depends on the conditions used for the precipitation. The irregular particle size was
observed at 1/3, regardless of pressure or temperature; especially in run 2 and 9, where
two types of particles were observed, probably from the PLE extract and PVP, so when a
1/3 ratio is used, an encapsulation of the antioxidant compounds is not achieved. This
effect can be seen in Fig. 4. When PVP is in defect it has been observed by other authors
[39-42, 56-57] that irregular shapes and no spherical particles were produced.
On the other hand, it is possible to observe that, if the content of PVP in the
solution is increased, the mean size of the precipitated particles increased and the particle
size distribution (D50) enlarged (Fig. 8). These results were coincident with the literature
[31]. For that reason, in this work, best morphology was obtained at 1/9 mass ratio,
preferably at pressures higher than 120 bar and between 35 and 65 ºC.
Finally, it must be noted that the presence of two or more solutes makes the
corresponding vapour liquid equilibrium diagram more complicated and, consequently, a
lower predictable precipitation. In some cases, it could be operated as a simple
precipitation according to the obtained results [31]. In this way nanoparticles would be
generated if the interfacial tension vanishing time is lower than jet break up time in a gas
to particle mechanism. Opposite, microparticles would be precipitated if there is any
residual interfacial tension after jet break-up. Thus expanded microparticles with a
continuous surface would be obtained when the diffusion is faster than nucleation stages.
In this case the precipitation is uniform throughout the droplet. However, if nucleation is
faster than diffusion stages, expanded particles with discontinuous structure are obtained.
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In that case the process is characterized by a diffusion limited precipitation front [58].
Anyway crystals with different shape and size can be produced when the operating
conditions are situated below mixture critical point, so in the two-phase region of the
vapor liquid equilibria diagram following a liquid-like crystallization mechanism. In this
case nuclei of compounds are formed directly in the liquid-phase and their growth is
produced by the migration of molecules from the liquid to surface of the crystal [31].
Anyway, not only the thermodynamic considerations but also hydrodynamic
considerations should be taken into account since flow and mixing conditions
significantly influenced on mass transfer kinetics so in the final feature of the precipitated
particles.
3.2. UPLC analysis
In this study, five antioxidants compounds have been identified and quantified in the
PLE precipitate. These compounds are: gallic acid, mangiferin, quercetin 3-D-galactoside, penta-
O-galloyl glucose and quercetin. In the antioxidants/PVP precipitates the quercetin could not be
quantified, due to the low concentration of the compound.
Penta-O-galloyl glucose and quercetin 3-D-galactoside were the major compounds as
can be observed in Table 2. As expected, in all experiments with a greater mass ratio, the amount
of antioxidant compounds was lower although all precipitates had antioxidant capacity. Previous
studies such as Huh Jet al. [59] and Bae et al. [60] have provided evidence about the strong
antiangiogenic and antiviral activity of penta-O-galloyl glucose , proposing as a potential
candidate antiviral drug to treat varicella-zoster virus associated diseases. On the other hand,
flavonoids as quercetin and quercetin derivatives as the quercetin 3-D-galactoside present
different benefits as anti-inflammatory, cardiovascular health, antibacterial and anticancer
effects.
3.3. In vitro release test
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The release of mango antioxidant/PVP particles, was studied focusing on the three
major compounds: mangiferin, quercetin 3-D-galactoside and penta-O-galloyl glucose. In
general, dissolution tests in both gastric and intestinal fluid confirmed the encapsulation of
mango antioxidants, due to showed a reduction of percentage released compared with
precipitates of mango leaves (PLE) without polymer. In Fig. 9 and 10, can be observed that time
of release of all experiments was slower than PLE precipitates alone. The antioxidants/PVP ratio
had a greater influence on the percentage of release, due to in all cases when mass ratio was
increased from 1/3 to 1/9 the release percentage was lower. These results could be due to the
different amount of PVP particles on the surface of microparticles of PLE precipitates as is shown
in Fig. 4, achieving a lower dissolution of the particles with greater amount of PVP [61]. In this
sense the diffusivity of particles, which were encapsulated, through the polymer would be lower
when polymer content is higher than when is lower producing a delay in the release.
On the other hand, the released percentage and dissolved of mangiferin in SIF was
slightly higher than in SGF, while that the opposite effect occurs with quercetin 3-D-galactoside.
In the case of penta-O-galloyl glucose, there was no difference in the release in both SIF and
SGF. These results can be due to the different solubility of compounds in both fluids [49,61].
With regard to the mangiferin release in both fluids, in the first thirty minutes around 50% of
this compound was released in both PLE precipitate and run 9, while in run 3 the release was
lower than 30%. At eight hours, the total release was higher than 60% in experiments 3 and 4
(1/9 mass ratio), whereas in runs 9 and 10 (1/3 mass ratio) it was between 80 and 90%. According
to release profile of quercetin 3-D-galactoside in gastric fluid, it can be observed that in run 4
(1/9) this compound releases slower than in other runs and in eight hours the percentage of
release was 50 % approximately. The release in gastric and intestinal fluid of penta-O-
galloyl glucose in SIF and SGF was higher in the 1/3 mass ratio runs, reaching a 100% of release
ACCEPTED MANUSCRIPT
in 8 hours. This mean that the experiments 9 and 10 are not totally encapsulated, but could be
co-precipitated [62].
Finally, the results of release profiles suppose that due to the successful encapsulation
of the antioxidant compounds of mango leaves in PVP, using 1/9 of mass ratio, a sustained
release of mangiferin, quercetin 3-D-galactoside and penta-O-galloyl glucose was achieved.
4. Conclusion
In this research, it was demonstrated that it is possible to obtain encapsulations of
mango leaves antioxidants in PVP using the SAE technique. In general, in all experiments
spherical or slightly agglomerated particles were obtained with particle size between 0.11-0.59
µm. Among the different conditions used in the process as a mass ratio, temperature and
pressure, the antioxidants/PVP mass ratio was the principal variable that affects (p>0.05) on
particle size and on the release profiles; indeed, increasing the ratio from 1/3 to 1/9 the particle
size increased and the percentage of release was lower, while that increasing the temperature
in the range of 35-65 ºC, the particle size increased and had no effect on the percentage of
release. Pressure was the variable that had the lowest effect on particle size and release profile.
The submicron particles contents in antioxidant compounds as mangiferin, acid gallic,
penta-O-galloyl glucose and quercetin 3-D-galactoside make them some excellent candidates to
be used for nutraceutical formulations. Furthermore, the dissolution test in the gastric and
intestinal fluid showed that the antioxidant particles alone dissolved faster than the
antioxidant/PVP systems; these results could be used as evidence of the effective control of PVP
for the sustained release of antioxidants from the mango leaves.
Acknowledgements
We gratefully acknowledge the AUIP for a PhD Studentship, the Spanish Ministry of
Science and Technology (Project CTQ2013-47058-R) and European Regional Development Fund
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(ERDF) for financial support, and Central Service of Science and Technology of University of Cádiz
for analyses.
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Fig. 1. SAS200 pilot plant; (B1) liquid extract pump, (B2) CO2 pump, (P1) precipitator vessel,
(S1) separator, (V1) automated back pressure regulator (V2) manual back pressure regulator
Fig. 2. The estimated effects of main and interaction factors on the antioxidants/PVP co-
precipitation
Fig. 3. Principal effect on particle size of mango leaves antioxidants/PVP co-precipitates
Fig. 4. SEM images of co-precipitates of mango leaves by SAE process using different
antioxidants/PVP mass ratio
Fig. 5. SEM images of co-precipitates of mango leaves by SAE process using different
temperature
Fig. 6. SEM images of co-precipitates of mango leaves by SAE process using different pressure
Fig. 7. SEM images of particles with different morphology of PLE, PVP and PLE/PVP
precipitates
Fig. 8. Particle size distribution of antioxidants/PVP particles precipitated at 120 bar, 35 ºC (run
4 and 9) and 65ºC (run 5 and 6)
Fig. 9. Dissolution profiles of mangiferin, quercetin 3-D-galactoside and penta-O-galloyl glucose
in pH 6.8
Fig. 10. Dissolution profiles of the mangiferin, quercetin 3-D-galactoside and penta-O-galloyl
glucose in pH 1.2
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Fig. 1. SAS200 pilot plant; (B1) liquid extract pump, (B2) CO2 pump, (P1) precipitator vessel,
(S1) separator, (V1) automated back pressure regulator (V2) manual back pressure regulator
B2
Extract
+
PVP
CO2 Tank
B1
P1
S1
V1
V2
CO2
Solvent
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Fig. 2. The estimated effects of main and interaction factors on the antioxidants/PVP co-
precipitation
0
0.5
1
1.5
2
2.5
3
3.5
Estimated effect
Variables
Antiox/PVP ratio (x1) Temperature (x2) x1x2 x2x3 Pressure (x3) x1x3
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Fig. 3. Principal effect on particle size of mango leaves antioxidants/PVP co-precipitates
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Run 4 (120 bar, 35ºC, 1:9)
Figure 3. PLE 1 and PVP
Fig. 4. SEM images of co-precipitates of mango leaves by SAE process using different
antioxidants/PVP mass ratio
Run 3
1/9 w/w
Run 10
1/3 w/w
180 bar- 65 °C
w/w
Run 4
1/9 w/w
Run 9
1/3 w/w
120 bar- 35 °C
Run 5
1/9 w/w
Run 6
1/3 w/w
120 bar- 65 °C
Run 7
1/9 w/w
Run 2
1/3 w/w
180 bar- 35 °C
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Fig. 5. SEM images of co-precipitates of mango leaves by SAE process using different
temperature
Run 3
65 °C
0.59µm
Run 7
35°C
0.12µm
180 bar- 1/9 w/w
Run 4
35 ºC
Run 5
65 ºc
Run 6
65 °C
0.21µm
Run 9
35 °C
0.11µm
120 bar- 1/3 w/w
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Fig. 6. SEM images of co-precipitates of mango leaves by SAE process using different pressure
Run 2
180 bar
Run 9
120 bar
35 °C - 1/3 w/w
Run 7
180 bar
Run 4
120 bar
35 ° C - 1/9 w/w
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Fig. 7. SEM images of particles with different morphology of PLE, PVP and PLE/PVP
precipitates
PLE
0.05 µm
PVP
0.57 µm
PLE + PVP
0.26 µm
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Fig. 8. Particle size distribution of antioxidants/PVP particles precipitated at 120 bar, 35 ºC (run
4 and 9) and 65ºC (run 5 and 6)
Run 9
Particle size (µm)
Density distribution
0 0,05 0,1 0,15 0,2 0,25 0,3
0
20
40
60
80 Distribución
Lognormal
Run 9
1/3 w/w
Run 5
Particle size (µm)
Density distribution
0 0,4 0,8 1,2 1,6
0
10
20
30
40
50
60 Distribución
Lognormal
Run 5
1/9 w/w
Run 6
Particle size (µm)
Density distribution
0 0,2 0,4 0,6 0,8
0
10
20
30
40
50 Distribución
Lognormal
Run 6
1/3 w/w
Run 4
Particle size (µm)
Density distribution
0 0,2 0,4 0,6 0,8 1
0
10
20
30
40
50 Distribución
Lognormal
Run 4
1/9 w/w
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Fig 9. Dissolution profiles of mangiferin, quercetin 3-D-galactoside and penta-O-galloyl
glucose in pH 6.8
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450
% Relaese Mangiferina
time (min)
Run 3 (1/9)
Run 4 (1/9)
Run 9 (1/3)
Run 10 (1/3)
Run 11 (1/6)
PLE precipitate
0
10
20
30
40
50
60
70
80
90
100
0100 200 300 400
% Release quercetin 3-D-galactoside
time (min)
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450
% Release penta-O-galloyl glucose
time (min)
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Fig 10. Dissolution profiles of the mangiferin, quercetin 3-D-galactoside and penta-O-galloyl
glucose in pH 1.2
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450
% Release Mangiferin
time (min)
Run 3 (1/9)
Run 4 (1/9)
Run 9 (1/3)
Run 10 (1/3)
Run 11 (1/6)
PLE precipitate
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450
% Release quercetin 3-D-galactoside
time (min)
0
10
20
30
40
50
60
70
80
90
100
050 100 150 200 250 300 350 400 450
% Release penta-o-galloryl glucose
time (min)
ACCEPTED MANUSCRIPT
Run order
P
bar
T
ºC
Extract/
PVP
Ratio
D50
µm
D10-D90
µm
Morphology
9
120
35
1/3
0.11±0.06
0.04 - 0.20
NP
4
120
35
1/9
0.33±0.05
0.19 - 0.50
MP
5
120
65
1/9
0.44±0.24
0.19 - 0.77
MP
6
120
65
1/3
0.21±0.13
0.09 - 0.36
MP
2
180
35
1/3
0.18±0.12
0.01 - 0.33
MP
7
180
35
1/9
0.12±0.06
0.08 - 0.17
NP
3
180
65
1/9
0.59±0.03
0.26 - 0.94
MP
10
180
65
1/3
0.13±0.13
0.06 - 0.20
MP
1
150
50
1/6
0.19±0.07
0.16 - 0.38
MP
8
150
50
1/6
0.26±0.07
0.18 - 0.35
MP
11
150
50
1/6
0.28±0.12
0.15 - 0.46
MP
12
150
50
1/9
0.26±0.11
0.13 - 0.41
MP
PVP
150
35
0/1
0.57±0.23
0.29 - 0.88
MP
PLE [53]
150
35
1/0
0.05±0.04
0.02 - 0.09
NP
MP: microparticles; NP: nanoparticles
Table 1. Conditions, mean particle sizes and size distributions of SAE processed particles
ACCEPTED MANUSCRIPT
Sample
Gallic
acid
Mangiferin
Quercetin 3-
D-
galactoside
Penta-O-
galloyl glucose
Quercetin
mg/g precipitate
PLE
precipitate
12.58
46.78
79.55
476.38
6.66
Run 6
1/3
4.27
15.75
29.64
126.22
NQ
Run 11
1/6
1.51
7.36
15.88
41.65
NQ
Run 3
1/9
1.82
5.48
11.05
36.91
NI
NQ: No quantified; NI: No identified
Table 2. Phenolic compounds in co-precipitates
ACCEPTED MANUSCRIPT
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