Molecules
Molecules
Molecules
Article
Design and Development of Spray-Dried
Microsystems to Improve Technological and
Functional Properties of Bioactive Compounds from
Hazelnut Shells
Tiziana Esposito, Teresa Mencherini * , Pasquale Del Gaudio , Giulia Auriemma ,
Silvia Franceschelli, Patrizia Picerno, Rita P. Aquino and Francesca Sansone *
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy;
[email protected] (T.E.); [email protected] (P.D.G.); [email protected] (G.A.); [email protected] (S.F.);
[email protected] (P.P.); [email protected] (R.P.A.)
* Correspondence: [email protected] (T.M.); [email protected] (F.S.); Tel.: +39-089-968294 (T.M.);
+39-089-968146 (F.S.)
Abstract: An extract obtained from hazelnut shells by-products (HSE) has antioxidant and
chemopreventive effects on human melanoma and cervical cancer cell lines, inducing apoptosis by
caspase-3 activation. A clinical translation is limited by poor water solubility and low bioavailability.
Dried plant extracts often show critical characteristics such as sticky/gummy appearance, unpleasant
smell, and instability involving practical difficulties in processing for industrial use. A spray drying
method has been applied to transform raw HSE in a microparticulate powder. The biopolymeric
matrix was based on l-proline as loading carrier, hydroxyethylcellulose in combination with pectin as
coating polymers; lecithin and ethanol were used as solubility enhancers. A Hot-Cold-Hot method was
selected to prepare the liquid feed. The thus prepared powder showed good technological properties
(solid-state, particle dimensions, morphology, and water dissolution rate), stability, and unchanged
chemopreventive effects with respect to the unprocessed HSE.
1. Introduction
By-products of agro-food manufacturing chains contain polysaccharides, proteins, vitamins, fats,
and phytochemicals. The recovery of bioactive components with health-promoting benefits suggests
the exploitation of vegetable residues as functional ingredients in developing nutraceuticals, functional
or novel foods, and pharmaceutical products [1–3]. Today, there is an increasing interest in the use of
nutraceuticals, based on plant extracts or food by-products, as potential chemopreventive agents, to be
used in combination with chemotherapeutics, as a new strategy in cancer control [4–6]. In previous
work, we have produced a polyphenol-rich extract (HSE) from the hazelnut shells, major by-product,
together with skins [7] of the kernel industrial processing. The neolignan lawsonicin was identified as
the HSE chemical marker. Moreover, the extract antioxidant and inhibitory effect inducing apoptosis
on human melanoma and cervical cancer cell growth were demonstrated [8]. Due to its functional
properties, the production of HSE represents an efficient way to re-use and up-cycle hazelnut shells,
turning these by-products, destined for landfill, into a resource of human health-promoting molecules.
However, as all dry plant derivatives, HSE appears as crystalline sticky material, not completely
soluble in water and aqueous biological fluids [9,10]. These critical features could limit the real use of
A good encapsulating agent (wall material) should have emulsifying and film-forming properties,
display low hygroscopicity, have low viscosity at high solid contents, be biodegradable, soluble
in aqueous solvents, bland in flavor/tasteless, low-cost, non-toxic, and food-grade [10]. However,
no single encapsulant possesses all these properties, and because of that, two or more encapsulants
must often be used in combinations. Pectin (PEC) is a structural component of the vegetable cell
wall. It is recovered from higher plants and widely used as a gelling agent, stabilizer, and thickener
in foods (E440). The polymer viscosity depends on the molecular weight, pH, ionic strength, and
arrangement of carboxyl groups [35,36]. PEC is a heterogeneous polysaccharide made up of linear
(1→4)-linked-α-d-galacturonic acid residues partially broken off by (1→2)-linked side chain consisting
of l-rhamnose and some other neutral sugar residues [37]. Pectin can be classified in low methoxylated
pectin (DE < 50%) and high methoxylated pectin (DE > 50%) according to their degree of esterification
(DE), expressed as a percentage of carboxyl groups [36,38]. High methoxylated pectin (DE > 50%) form
a particularly stable gel polymeric network, both at acid pH (pH ≈ 5) and in the presence of sugars,
without adding divalent ions as occurs for pectin with a low DE or alginates. Moreover, the high
methoxylated pectin is characterized by a low hygroscopicity; an important property to improve
product stability during storage [39].
The lecithin (L) molecule is a food additive (E322) used as an excellent natural emulsifier, stabilizer,
and dispersing agent. The amphiphilic properties allow them to increase the water affinity of non-polar
ingredients (fats), arranging the hydrophobic portion around them, and the hydrophilic moiety in the
aqueous phase [40].
The aim of the present research was the production of new polymeric microparticulate HSE-based
powders by spray drying technique. The appropriate polymeric matrix, as coating polymers and
loading carriers, able to transform HSE in a microparticulate powder form, was selected. The influence
of instrumental and operating parameters on yield and encapsulation efficiency was evaluated.
The produced microparticulate powders were characterized for solid-state (particle dimensions,
morphology, amorphous or crystalline form), and water dissolution rate. The optimized formulation
was subjected to accelerated stability tests under harsh storage conditions (ICH-International Conference
on Harmonization). Finally, the functional stability of the HSE-loaded particle system was investigated.
Preliminary satisfactory results in terms of matrix processability were obtained with an HEC and
P concentration of 0.2% and 5.0% w/v (Batch-3), respectively. Normally, the morphology of P shows a
needle crystalline form [25] (Figure 1a) and the unprocessed extract appeared as a material in a cluster
form with irregular shape and surface (Figure 1b) and exploiting red and yellow fluorescence due to
the different nature of polyphenols present in the extract (Figure 1b).
Spray-dried Batch-3 showed a P interaction with HEC, involving in a spherical agglomeration
process, without totally losing its crystalline state (Figure 1c) [42] causing fractures and clusters on
particles surface. Moreover, the loading with HSE further negatively affected the agglomeration
process of the matrix system (Batch 13, Figure 1d). After the spray drying process, P, in combination
with HEC and HSE, did not lose its crystalline state, leading to an irregular and not homogeneous
solid-state Batch 13, Figure 1d).
With the aim to both promote the particle formation and reduce the crystalline surface, also
increasing process yield, high methoxylated pectin from citrus (PEC) was selected as an additional
coating polymer, separately included in the basic polymeric matrix. The presence of crystals can
fracture the outer surface of the particles and promote the release of the extract. This is a cause
of instability because the extract was exposed and, therefore, subject to degradation. In previous
work, PEC has proved suitable to stabilize maltodextrins as a carrier of sticky plant extracts [35].
The percentage of PEC was tested in formulation from 0.25 to 1% w/v. The resultant strategy was
best effective (yield 45.0%) at the 0.5% PEC concentration (Batch-9, Figure 1e, Table 1). The highest
concentrations (1.0% w/v) of PEC, in combination with HEC, led to a drastic reduction in process
yield (15.6%), probably caused by an increase in the viscosity of the matrix. As shown in Figure 1e,
the addition of PEC as coating copolymer improved the spherization of the particles, and no aggregates
were detected.
Once the polymeric matrix conditions were established, the next step was to load the HSE extract.
Lecithin (L) as emulsifier dissolved in ethanol (EtOH) [43] was added to the liquid feed preparation
to overcome the critical solubility characteristics of the plant extract. Moreover, a “Hot-Cold-Hot”
(H-C-H) method preparation was used. The formulation of a multi-component matrix requires,
for reasons linked to the different characteristics of the ingredients, a multi-step preparation method.
The Hot-Cold-Hot method depends on the solubilization conditions of HEC that need heating to
dissolve in water. Subsequently, the extract needs a lower temperature because it is a thermosensitive
substance. The thus prepared liquid feed reflected in the best-obtained result in terms of process
yield (50%) and particle morphology (Batch-12, Figure 1f) for unloaded powders. The best HSE
concentration, compatible with the developed polymeric system, resulted in the 0.25% w/v (Batch-15,
yield 43.0%, Table 1), with respect 0.50% w/v (Batch-13, yield 39.8%, Table 1).
The actual extract content (AEC-HSE) of Batch-15 resulted very close to the amount of extract used to
prepare the liquid feed (theoretical extract content, TEC-HSE). Consequently, the loading efficiency (LE)
Molecules 2020, 25, 1273 5 of 19
value, calculated as the ratio of AEC to TEC, was very satisfying (95.12%, Table 1). However, for optimized
powders, the process yield did not exceed 50% probably for the low amount of material sprayed (100 mL)
Molecules 2020, 25, x FOR PEER REVIEW 6 of 20
and the loss of the smallest and lightest particles with the exhaust of the spray dryer [15].
Figure 1. Fluorescence (FM) images in bright field (a) P, proline spray drying, (b) hazelnut shells by-
Figure 1. Fluorescence (FM) images in bright field (a) P, proline spray drying, (b) hazelnut shells
products (HSE) raw material in crystalline form; scanning electron microscopy (SEM) micrographs
by-products (HSE) raw material in crystalline form; scanning electron microscopy (SEM) micrographs
(c) Batch-3; (d) Batch-13, (e) Batch-9, (f) Batch-12, (g) Batch-15, FM image (h) Batch-15.
(c) Batch-3; (d) Batch-13, (e) Batch-9, (f) Batch-12, (g) Batch-15, FM image (h) Batch-15.
Molecules 2020, 25, 1273 6 of 19
Morphological analysis showed that for Batch-15 (Figure 1g), spherical, well-formed,
and completely coated microparticles had been obtained during the spray drying process. Furthermore,
the extract was homogeneously distributed and better encapsulated within the microparticles
(Figure 1g,h).
Molecules 2020, 25,In fact,PEER
x FOR Batch-15
REVIEWmicroparticles showed a pale-yellow fluorescent (Figure 1h), which was
7 of 20
the result of the combination between red/yellow-HSE and blue blank powder as evidence of interaction
in forming a homogeneous matrix.
As reported
2.2. Thermal Analyses in Table 1, Batch-13 and Batch-15 microparticles also had different size distribution
(d50 18.41 and 3.0 µm) with the lowest mean diameter observed for Batch-15 (Table 1). This difference
was To
dueprovide
to the rightinformation on solid-state
physical interaction and matrix
between extract-polymer
componentsinteractions, as rearrangement
leading to the well as on the of
physical stability of materials after the technological process, differential scanning
P crystals in spherical agglomerates during the spray drying process. The use of the organic calorimetric
lecithin
technique
solution towas used
better [44]. To
expose the evaluate
extract tothe
thephysical
aqueousinteraction
polymeric after the formation
feed seemed of the affect
to positively particles
the
during the spray drying process, the thermal trends by DSC of all raw materials with respect
interaction of extract with the matrix components, resulting in the reduced dimensional distribution to the of
spray-dried
the obtainedformulations
particles. were shown (Figures 2 and 3).
The heating curve of the HEC raw material showed a slight endothermic event at the baseline
above 100–120
2.2. Thermal °C, due to the dehydration phase. The water loss up to 120 °C was generally followed
Analyses
by a two-step thermal event of cellulose, depolymerization, and decomposition [45]. In the
To provide information on solid-state and extract-polymer interactions, as well as on the physical
thermogram (Figure 2, blue line), only an exothermic transition was visible (at around 310 °C), which
stability of materials after the technological process, differential scanning calorimetric technique
may be related to the depolymerization of cellulose material. This event anticipated the pyrolytic
was used [44]. To evaluate the physical interaction after the formation of the particles during the
decomposition [46], which could occur in a 400 to 470 °C range of temperature, and was not detected
spray drying process, the thermal trends by DSC of all raw materials with respect to the spray-dried
here. The proline raw material showed a first endothermic peak at 217 °C with the final melting peak
formulations were shown (Figures 2 and 3).
at 246 °C (Figure 2, black line).
Differentialscanning
Figure2.2.Differential
Figure scanningcalorimetry
calorimetry(DSC)
(DSC)ofofraw
rawmaterials:
materials:HEC,
HEC,blue
blueline;
line;PEC,
PEC,red
redline;
line;P,P,
black line; HSE extract raw material, green line.
black line; HSE extract raw material, green line.
The heating curve of the HEC raw material showed a slight endothermic event at the baseline
The thermal profile by DSC of the unprocessed HSE raw extract (Figure 2, green line) exhibited
above 100–120 ◦ C, due to the dehydration phase. The water loss up to 120 ◦ C was generally followed by
a series of endothermic events due to the melting of the active components, mainly polyphenols, in a
a two-step thermal event of cellulose, depolymerization, and decomposition [45]. In the thermogram
range of temperatures between 130 and 250 °C. As for other natural extracts, it was not possible, for
(Figure 2, blue line), only an exothermic transition was visible (at around 310 ◦ C), which may be related
HSE, to see a single melting peak as for pure components [17,18,35].
Figure 3 shows the thermal behavior of the spray-dried powders. The first endothermic event,
at baseline, in the temperature range of 50 to 100 °C, was due to free water loss. As shown by the
thermogram (Figure 3), the amount of residual water was lower for Batch-15 with respect to Batch-3,
and Batch-13 could positively affect the stability of Batch-15 powder.
Comparing DSC thermal profiles of Batch-12 and Batch-3 (blank powders, Figures 3, dotted
Molecules 2020, 25, 1273 7 of 19
to the depolymerization of cellulose material. This event anticipated the pyrolytic decomposition [46],
which could occur in a 400 to 470 ◦ C range of temperature, and was not detected here. The proline raw
material showed a first endothermic peak at 217 ◦ C with the final melting peak at 246 ◦ C (Figure 2,
black line).
The thermal profile by DSC of the unprocessed HSE raw extract (Figure 2, green line) exhibited
a series of endothermic events due to the melting of the active components, mainly polyphenols, in
a range of temperatures between 130 and 250 ◦ C. As for other natural extracts, it was not possible,
for HSE, to see a single melting peak as for pure components [17,18,35].
Figure 3 shows the thermal behavior of the spray-dried powders. The first endothermic event,
at baseline, in the temperature range of 50 to 100 ◦ C, was due to free water loss. As shown by the
thermogram (Figure 3), the amount of residual water was lower for Batch-15 with respect to Batch-3,
and Batch-13 could positively affect the stability of Batch-15 powder.
Molecules 2020, 25, x FOR PEER REVIEW 8 of 20
Comparing DSC thermal profiles of Batch-12 and Batch-3 (blank powders, Figure 3, dotted black
and red line, respectively), proline showed an anticipation of melting peak probably due to the effect
of agglomeration on P crystals [31,42], shifted by 8 Celsius degrees in Batch-15 (221.45 ◦ C) with
respect to Batch-13 (228.81 °C). This event was accompanied by a reduction of enthalpy, indicating a
respect to Batch-13 (228.81 ◦ C). This event was accompanied by a reduction of enthalpy, indicating
reduction in crystallinity. Even if there was a melting peak attributable to a crystalline residue of
a reduction in crystallinity. Even if there was a melting peak attributable to a crystalline residue
proline, the produced systems could be defined as an amorphous multicomponent ingredient. The
of proline, the produced systems could be defined as an amorphous multicomponent ingredient.
structure of amorphous solids was not random at the molecular level but may possess short-range
The structure of amorphous solids was not random at the molecular level but may possess short-range
order, residual crystallinity, polymorphic states, and regions of different density. Many
order, residual crystallinity, polymorphic states, and regions of different density. Many pharmaceutical
pharmaceutical formulations (multi-component systems) are formed by either single or multiple
formulations (multi-component systems) are formed by either single or multiple active substances and
active substances and drug excipients. One or more of the components can be present in the
drug excipients. One or more of the components can be present in the amorphous state [47].
amorphous state [47].
Finally, in all thermograms, it was possible to observe a thermal event around 290 ◦ C, due to the
Finally, in all thermograms, it was possible to observe a thermal event around 290 °C, due to the
final product degradation. This event possessed a lower intensity for Batch-15 (Figure 3) for Batch-13
final product degradation. This event possessed a lower intensity for Batch-15 (Figure 3) for Batch-13
(Figure 3), indicating that the extract was completely encapsulated in the matrix. No new peaks
(Figure 3), indicating that the extract was completely encapsulated in the matrix. No new peaks
ascribable to chemical interactions were detected.
ascribable to chemical interactions were detected.
The thermal analysis of the optimized formulation Batch-15 confirmed that extract well interacts
The thermal analysis of the optimized formulation Batch-15 confirmed that extract well interacts
in forming microparticles with the polymeric matrix. Moreover, DSC confirmed SEM (Figure 1g)
in forming microparticles with the polymeric matrix. Moreover, DSC confirmed SEM (Figure 1g)
results that showed microparticles well-formed with a reduction of proline in a crystalline state, also
results that showed microparticles well-formed with a reduction of proline in a crystalline state, also
supposing a consequent better behavior instability [20,35].
supposing a consequent better behavior instability [20,35].
Differentialscanning
Figure3.3.Differential
Figure scanningcalorimetry
calorimetry(DSC)
(DSC)of
ofBatch-12,
Batch-12,Batch-15,
Batch-15,Batch-3,
Batch-3,and
and Batch-13.
Batch-13.
Figure
Figure 4. Dissolution/release profile of Batch-13 and Batch-15
4. Dissolution/release Batch-15 microparticles compared with HSE
HSE
(unprocessed
(unprocessed extract).
extract).
humidity content. Figure 5 shows the thermal profiles of Batch-15 after a storage period of 1 month
(black line) with respect to Batch-15 at t0 (48h in a desiccator, gray line). The results displayed the
presence of a small percentage of free water, absorbed during the spray drying process, after 48 h in
a desiccator. The peak was already visible after 1 month (Figure 5, gray and black lines). However,
Molecules 2020, 25, x FOR PEER REVIEW 10 of 20
the presence
Molecules of water,
2020, 25, onlyREVIEW
x FOR PEER affected the morphology of particles without causing no degradation10events
of 20
or formation of chemical interactions (Figure 5) because no new peaks have been detected.
Figure
Figure 5. Differential scanning
5. Differential scanning calorimetry
calorimetry(DSC)
(DSC)thermograms
thermogramsofofBatch-15
Batch-15(48 desiccator)atatt0,t0,
(48hhinina adesiccator) 0,
and
and after
after 1 month.
In
In fact,
In fact, in the
fact, in the SEM images,
SEM images,
images, nono breakings
no breakings werevisible
breakings were
were visible inthe
visiblein
in the microstructures,
themicrostructures, althougha aahigh
microstructures,although
although high
high
degree
degree
degree ofof aggregates and
of aggregates and particles
particles showed
particles showed an
showed an irregular
an irregularshape
irregular shapeand
shape andsurface
and surfacewith
surface withthe
with thepredominant
the predominant
predominant
crystalline
crystalline aspect evident (Figure
crystalline aspect evident (Figure 6 and
(Figure 66 and b).
and b).
b).
Figure 6.
Figure 6. Scanning
Scanning electron microscopy (SEM) images of Batch-15 (48 hhininaadesiccator)
desiccator)atat
att0tt(a) and
Figure 6. Scanning electron
electron microscopy
microscopy (SEM)
(SEM) images
images of
of Batch-15
Batch-15 (48
(48 h in a desiccator) 0 (a) and
0 (a) and
after
after 1 month (b).
after 11 month
month (b).
(b).
The physical
The physical instability
instability
instability was
was probably
was probably due
probably due toto the
the residual
residual process
processhumidity
humiditycontent
contentofofthe
the
powder. Hygroscopicity
powder. Hygroscopicity results indicated that during the harsh storage conditions, a water loss
powder. Hygroscopicityresults
resultsindicated that
indicated during
that duringthe harsh storage
the harsh conditions,
storage a water
conditions, a loss
wateroccurs,
loss
occurs, gravimetrically determined, of 2.40 g/100 g ± 0.82. The mobility of this amount of residual
gravimetrically
occurs, determined,
gravimetrically of 2.40 g/100
determined, g ±g/100
of 2.40 0.82. gThe mobility
± 0.82. The of this amount
mobility of thisofamount
residualof
free-water
residual
free-water was responsible for morphology changes.
was responsible
free-water for morphology
was responsible changes. changes.
for morphology
Thus, the storage period of Batch-15 in desiccator was extended to 72 h, and the accelerated
Thus, the storage period of Batch-15 in desiccator was extended to 72 h, and the accelerated
stability assay was repeated, in the same condition. The results obtained exhibited a clear reduction
stability assay was repeated, in the same condition. The results obtained exhibited a clear reduction
of the free water (gravimetrically quantified, 0.41 g/100g ± 0.03) (Figure 7), and the morphology of
of the free water (gravimetrically quantified, 0.41 g/100g ± 0.03) (Figure 7), and the morphology of
the particles resulted unaltered, also after 6 months (Figure 8 a,b).
the particles resulted unaltered, also after 6 months (Figure 8 a,b).
Molecules 2020, 25, 1273 10 of 19
Thus, the storage period of Batch-15 in desiccator was extended to 72 h, and the accelerated
stability assay was repeated, in the same condition. The results obtained exhibited a clear reduction of
the free water (gravimetrically quantified, 0.41 g/100g ± 0.03) (Figure 7), and the morphology of the
Molecules 2020, 25, x FOR PEER REVIEW 11 of 20
particles resulted
Molecules 2020, unaltered,
25, x FOR also after 6 months (Figure 8 a,b).
PEER REVIEW 11 of 20
Figure
Figure7.7.
Figure Differential
7.Differential scanning
Differentialscanning
scanning calorimetry
calorimetry
calorimetry (DSC)
(DSC) thermograms
thermograms
thermograms of Batch-15
ofofBatch-15
Batch-15(72 (72
(72h hin ah desiccator)
ina desiccator) t0, t0, at t0,
in a desiccator)
at at
and
and
and after 6 months.
after6 6months.
after months.
Figure8.
Figure 8. Scanning
Scanning electron
electronmicroscopy
microscopy(SEM) images
(SEM) of Batch-15
images (72 h (72
of Batch-15 in ahdesiccator) at t0 (a)at
in a desiccator) and
t0 (a) and
Figure
after 68.months
Scanning
(b).electron microscopy (SEM) images of Batch-15 (72 h in a desiccator) at t0 (a) and
after 6 months (b).
after 6 months (b).
The lawsonicin content, the major compound of HSE, and the free radical scavenging activity
The lawsonicin content, the major compound of HSE, and the free radical scavenging activity
wereThe lawsonicin
verified, both content,
after the the
spraymajor
dryingcompound of HSE,
process (after 72 hand the desiccator)
in the free radicaland scavenging activity
after 6 months
were verified,
were
verified, both
both after
after
the spray drying
the spray
process (after 72 h in the desiccator) and after 66months under
under the storage condition and drying
compared process
with(after 72 h in the desiccator)
the unprocessed extract (HSE).and after months
The results,
the
understorage condition
the storage
summarized in Table and
condition compared
2, showedand with
thatcompared the
Batch-15 didwithunprocessed extract
the unprocessed
not exhibit (HSE). The
extract (HSE).
a significant reduction results, summarized
The results,
of the lawsonicin in
Table 2,
summarized showed
content (lower that
in Table Batch-15
2, showed
than 1%) after thedid
that not exhibit
Batch-15 did not
transformation a significant
exhibit
process reduction
anda6significant of the
months ofreduction lawsonicin
storage. On of the content
the contrary,
lawsonicin (lower
than
HSE1%)
content provedaftersignificantly
(lower the transformation
than 1%) after(p the
< 0.05) process andprocess
lower active
transformation 6content.
months of storage.
Moreover,
and 6 months ofOn
the the contrary,
antiradical
storage. Onactivity HSE
was proved
the contrary,
significantly
evaluated by(p <
the 0.05)
DPPH lower
test to active
verify content.
that the Moreover,
formulation the antiradical
HSE proved significantly (p < 0.05) lower active content. Moreover, the antiradical activitynot
process and the activity
storage was
period evaluated
have wasby the
DPPH testby
altered
evaluated theto the
verify
free DPPHthattest
radical the to
formulation
scavengingverify thatprocess
activity of HSE and the storage
[35].
the formulation As processperiod
reported in have
Table
and the 2, not altered
the
storage periodthe
effect free
remained
have radical
not
unaltered
scavenging
altered after
the free the spray
activity
radical drying
[35].process
of scavenging
HSE As (ECof
reported
activity 50 33.20 ±[35].
0.61 µg/mL)
in Table
HSE 2, the
As and until
effect
reported 6 months
remained
in Table ofeffect
harsh after
2,unaltered
the storage
the spray
remained
conditions (EC 50 32.80 ± 1.01 µg/mL). On the contrary, at the same conditions, HSE raw extracts
± 0.61
drying
unaltered process
after (EC
the 50 33.20
spray drying process
µg/mL) (ECand until
50 33.20 6 months
± 0.61 µg/mL) ofand
harsh
untilstorage
6 months conditions
of harsh(EC 50 32.80 ±
storage
proved a significant
conditions (EC50 32.80(p±< 1.01
0.05) µg/mL).
reductionOn of EC
the50 values
contrary, fromat33.42 ± 1.40 µg/mL
the same to 40.04
conditions, HSE± 2.11
rawµg/mL.
extracts
proved a significant (p < 0.05) reduction of EC50 values from 33.42 ± 1.40 µg/mL to 40.04 ± 2.11 µg/mL.
Molecules 2020, 25, 1273 11 of 19
1.01 µg/mL). On the contrary, at the same conditions, HSE raw extracts proved a significant (p < 0.05)
reduction of EC50 values from 33.42 ± 1.40 µg/mL to 40.04 ± 2.11 µg/mL.
Table 2. Actual Active Content (%) and free-radical scavenging activity of extract before (HSE
unprocessed extract) and after microencapsulation process (Batch-15).
0 6 Months 0 6 Months
Materials AAC% a,b DPPH test EC50 a,c,d
HSE Unprocessed extract 3.16 ± 0.80 1.15 ± 0.40 * 33.42 ± 1.40 40.04 ± 2.11 *
Batch-15 2.10 ± 0.40 2.00 ± 0.60 33.20 ± 0.61 32.80 ±1.01
α-tocopherol e 10.1 ± 1.32 10.12 ± 1.20
aOne-way analysis of variance (ANOVA) followed by and Tukey HSD test; means ± SD, * p <0.05; b Actual Active
Content (AAC): Content of lawsonicin calculated by HPLC-DAD; c EC50 ± standard deviation (data from three
experiments in triplicate); d In a unit of µg of HSE or Batch-15/mL; e Positive control of the DPPH test
The results demonstrated that the optimized matrix and process parameters allowed to protect
the antiradical efficacy of HSE, obtaining a long-lasting, stable microparticulate system.
Table 3. MTT assay of raw extract (HSE) and optimized batch (Batch-15).
3.1. Chemicals
Analytical grade methanol (MeOH), HPLC-grade methanol (MeOH), isopropanol, ethanol (EtOH),
1,1-diphenyl-2-picrylhydrazyl radical (DPPH), α-tocopherol, and l-proline (P) were obtained from
Sigma-Aldrich (Milan, Lombardia, Italy). Medium viscosity hydroxyethylcellulose (HEC, Natrosol
MR) and lecithin (L, E322) were supplied by ACEF Spa (Fiorenzuola D’Arda, PC, Italy). Pectin (PEC
09/051, 76% degree of esterification) was recovered from the citrus peel obtained during the processing
of Agrumi Gel s.n.c. Company (Barcellona Pozzo DI Gotto, Messina, Italy). HPLC-grade water (18 mΩ)
was prepared by a Milli-Q50 purification system (Millipore Corp., Bedford, MA, USA). The hazelnut
shells extract (HSE) was prepared with the extraction method previously reported [8].
- Hot-Cold-Hot method (H-C-H): The liquid feed was produced using 0.2% w/v HEC, 5% w/v P
and 0.2% L at 85.0:3.4:8.2:3.4 P:HEC:PEC:L ratio (total amount 6.15 g). PEC was added in 80 mL
of water at 75 ◦ C; at room temperature P was included, and finally, at 50 ◦ C, HEC was dissolved,
leaving under stirring overnight. Separately, 0.25% or 0.50% w/v of HSE was dissolved adding
20 mL of ethanol and 20 mL of a 1% w/v L solution by homogenization with an Ultra-Turrax
T-25 (IKA ULTRA-TURRAX T25 digital) at 10,000 RPM for 5 min. The suspension containing the
extract (HSE) was slowly poured into the feed under continuous magnetic stirring.
All the liquid feeds were spray-dried using a Büchi B-191 Mini Spray Dryer (Büchi
Laboratoriums-Technik, Flawil, Switzerland). The Mini Spray Dryer operates according to a co-current
drying gas (e. g. air in open mode) and product stream. This means that sprayed products and hot gas
have the same flow direction from downward.
The experimental conditions were drying airflow 600 L/h, air pressure 6.5 bar, aspirator 100%
(maximum gas flow rate of about 35 m3 /h), inlet/outlet temperatures 125/85 ◦ C, Ø nozzle = 0.7 mm,
spray flow feed rate 3.8 mL/min.
Each preparation was produced in triplicate. Before characterization, the powders were stored in
the desiccator (48–72 h).
Molecules 2020, 25, 1273 13 of 19
The extract encapsulation efficiency (EE%) was the ratio of the actual to the theoretical extract
content: [13]:
EE% = AEC/TEC × 100 (2)
Each analysis was made in triplicate and results expressed as an average value.
volume of methanol. α-tocopherol was used as a positive control. The DPPH concentration in the
reaction medium was calculated from a calibration curve (range = 5–36 µg/mL) analyzed by linear
regression (y = 0.0228x − 0.0350, R2 = 0.9999), and EC50 (mean effective scavenging concentration) was
determined as the concentration (in micrograms per milliliter) of sample necessary to decrease the
initial DPPH concentration by 50%. All tests were performed in triplicate. A lower EC50 value indicates
a stronger antioxidant activity.
Hygroscopicity
The hygroscopicity was determined according to the procedure described by Sansone et al. [20]
by sampling during the end of the stability test.
Hygroscopicity was gravimetrically calculated according to the formula:
where m0 and m3 express the moisture of the samples before (t0 ) and after (t6months ) the storage
period, respectively.
4. Conclusions
Hazelnut shells extract has antioxidant and chemopreventive effects on human melanoma and
cervical cancer cell lines. However, as all dry plant derivatives, HSE appears as a sticky material,
not completely soluble in water and aqueous biological fluids, as well as quite unstable under
environmental stress. Our research showed the possibility to successfully transform the raw extract
HSE in a spray-dried powder suitable for the final administration in various dosage forms overcoming
Molecules 2020, 25, 1273 16 of 19
the stability and bioavailability problems. The tandem polymeric matrix was based on l-proline as a
synergic loading carrier, hydroxyethylcellulose/pectin as a coating system, and lecithin as an enhancer
of dissolution rate. The development of a multicomponent polymeric matrix, the optimization of the
liquid feed preparation method, and the selection of all the spray drying operating parameters led to
obtain a stable powder (Batch-15) with high functional content, and both improved water solubility
and chemopreventive effects against human cancer cells (A375, SK-Mel-28, and HeLa), easier to be
processed for an industrial use with respect to the unprocessed HSE.
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Sample Availability: Samples of the raw extract (HSE) and Spray-dried powder are available from the authors.
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