Received: 21 March 2017 | Revised: 20 June 2017 | Accepted: 9 July 2017

DOI: 10.1111/jfpp.13446

ORIGINAL ARTICLE

Formulation of stable microcapsules suspensions content Salvia

officinalis extract for its antioxidant activity preservation

Yacine Nait Bachir1 | Amel Zafour1 | Meriem Medjkane2

1
Chemical Engineering Laboratory, Process
Engineering Department, Faculty of
Technology, University of Saad Dahlab-Blida
1, Blida, Algeria
2
Laboratory of Natural Bio-Resources,
Department of Biology, Faculty of Science,
Hassiba Benbouali University of Chlef, Chlef,
Algeria
Correspondence
Yacine Nait Bachir, Chemical Engineering
Laboratory, Process Engineering
Department, Faculty of Technology,
University of Saad Dahlab-Blida 1, Algeria.
Email: phd.nait.bachir.yacine@gmail.com
Abstract
The stabilization of microparticles suspensions is always assured by macromolecules that allow
the increasing of dispersing phase viscosity and maintaining the microparticles dispersibility. In
this work, the effect of xanthan gum, tragacanth, and sodium alginate on the stability of gelatin–
pectin microcapsules suspensions containing sage polyphenols as an active substance was inves-
tigated. After polyphenols extraction from Salvia officinalis, their characterization by HPLC-UV/
DAD and their microencapsulation by complex coacervation technique using gelatin and pectin.
Three different suspensions of microcapsules were prepared using different polymeric dispers-
ant agents. The suspensions were characterized by laser for particle size, zetametry,
viscosimetry, and evaluation of their antioxidant activities was carried out by the DPPH scav-
enging radical method. Stability study of prepared suspensions was undertaken during 90 days,
the results obtained showed that sodium alginate and tragacanth had a better stabilizing effect
compared with xanthan gum. After formulation of sage extract, its antioxidant activity increases
and its half-life time increases from 12.75 6 1.95 days (R2 5 .93) to 258.64 6 21.99 days
(R2 5 0.98).

Practical applications
Microencapsulation yield of sage extract in gelatin–pectin is 73.54 6 2.04%. About 0.5% of sodium
alginate permits the stabilization of microcapsules suspension. Sodium alginate and tragacanth had
a better stabilizing effect compared with xanthan. After formulation, antioxidant activity of sage
extract increases. After formulation, half-life time of sage extract increases from 12.75 6 1.95 to
258.64 6 21.99 days.

1 | INTRODUCTION
The stabilization of suspensions containing microparticles is a purpose
that different industries look to achieve. Effectively, several active
ingredients used in different industries are insoluble in aqueous solu-
tions and present a very low physicochemical stability in these environ-
ments, as some drugs (Blessy, Patel, Prajapati, & Agrawal, 2014),
preservatives (De Vos, Faas, Spasojevic, & Sikkema, 2010), antioxidants
(Hern
andez-Jaimes, Fouconnier, Pe

rez-Alonso, Munguía-Guille

n, &
Vernon-Carter, 2013), pesticides (Szente, 1998), bacteria (Muhammad,
Ramzan, Huo, Tian, & Bian, 2017), and chemical catalysts (Scott, Datye,
& Crooks, 2003), used by pharmaceutical, food, cosmetics, phytosani-
tary, and chemical industries, respectively.
Physical instability of these suspensions is described by a rapid
separation of the dispersed phase and a very difficult redispersibility
of this phase in continuous phase (Vincent, 1974). On the other hand,
the chemical instability is mainly due to the effect of water compos-
ing the dispersing phase on different dispersed ingredients (Di Mattia,
Sacchetti, Mastrocola, & Pittia, 2009).
Xanthan gum is a natural polysaccharide of microbial origin that
presents high viscosifying effect (Garcıa-Ochoa, Santos, Casas, &
Gomez, 2000). Tragacanth is a natural gum composed of two differ-
ent types of polysaccharides, tragacanthine (30–40%) which is a
water-soluble hydrocolloid and bassorin (60–70%) which is insoluble
in water and swells for forming a hydrogel (Azarikia & Abbasi, 2010).
Alginates are natural polysaccharides extracted from marine algae
having a high negative charge (George & Abraham, 2006). The com-
mon point of these three different polymers previously described
(xanthan gum, tragacanth, and sodium alginate) is their ability to
increase the viscosity of an aqueous solutions (Azarikia & Abbasi,

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https://doi.org/10.1111/jfpp.13446
2 of 10 | NAIT BACHIR
2010; Garcıa-Ochoa et al., 2000; George & Abraham, 2006; Taher-
ian, Fustier, Britten, & Ramaswamy, 2008), which allows them to be
used as dispersing agents.
Salvia officinalis is a plant of Mediterranean origin and member of
the mint family Lamiaceae, it is traditionally used in cooking, infusion
preparation, and aromatherapy.
This aromatic plant containing essential oil and polyphenols is one
of the most widely plants used in cosmetics (Silvestin et al., 2012),
pharmaceutical (Hamidpour, Hamidpour, Hamidpour, & Shahlari, 2014),
and food industries (Hayouni et al., 2008).
The problem encountered with these bioactive molecules
extracted from sage and other plants is their great sensitivity to envi-
ronmental conditions like light, temperature, humidity, and oxygen
(Desai & Jin Park, 2005). Effectively, according to a study reported by
€hlinger, & Monakhova, 2012) the pol-
(Walch, Lachenmeier, Kuballa, Stu
yphenols of sage infusion are completely reduced after 60 hr of stor-
age, this is why we have chosen this plant as a model to study the
preservation of the bioactive molecules extracted from plants.
The originality of the present work is to study the effect of differ-
ent polymeric dispersing agents on the physicochemical stability of an
active ingredient which has been previously encapsulated in a poly-
meric membrane. At first, bioactive molecules are extracted from Salvia
officinalis, characterized by HPLC-UV/DAD, and then encapsulated by
complex coacervation technique using gelatin (polycation) and pectin
(polyanion). After that, the suspensions based on these microcapsules
are prepared using different dispersing agents (xanthan gum, traga-
canth, and sodium alginate), the suspensions will be characterized by
laser particle size, zetametry, viscosimetry, and their antioxidant activ-
ities evaluation will be made by DPPH scavenging radical method.
Finally, physicochemical and antioxidant properties of different suspen-
sions will be evaluated during 90 days of storage and suspensions sta-
bilization mechanisms will be proposed for each dispersing agent.
2 | MATERIALS AND METHODS
2.1 | Materials
Xanthan gum (Rhodia, Algiers, Algeria), Gelatin type B from bovine skin
(Sigma Aldrich, Taufkirchen, Germany), and sodium alginate (Sigma
Aldrich, Deisenhofen, Germany) were provided by the pharmaceutical
group Saidal (Algiers, Algeria). Tragacanth fine powder (Merck, Darm-
stadt, Germany), pectin from Citrus fruits (Sigma Aldrich, Taufkirchen,
Germany), Folin–Ciocalteu (Sigma Aldrich, Taufkirchen, Germany),
diphenyl-picryl-hydrazyl DPPH (Sigma Aldrich, Taufkirchen, Germany),
and gallic acid 1-hydrate (Panreac quimica, Barcelona, Spain) were
purchased.
HPLC standards: caffeic acid, ferulic acid, rutin, rosmarinic acid,
and quercitrin were purchased from Sigma Aldrich (St. Louis, MO,
USA). Formaldehyde, acetic acid, methanol, ethanol, phosphoric acid,
acetonitrile, sodium hydroxide, and sodium carbonate were obtained
from Biochem chemopharma (Cosne-Cours-sur-Loire, France). All
chemical products and distilled water were of analytical grade.
ET AL.
2.2 | Preparation and characterization of sage extract
2.2.1 | Preparation of extract
The extraction was performed by a Soxhlet extractor (Riviera, 500 ml
capacity, Mumbai, India) using ethanol as extraction solvent. About
50 g of plant powder was introduced into a cellulose extraction thim-
ble, the thimble was placed in a Soxhlet, equipped with a 1 L distillation
flask containing 750 ml of ethanol. Heating was provided with a heat-
ing mantle (Electrothermal, Staffordshire, UK). After 120 min of extrac-
tion, the extract was recovered after solvent evaporation.
2.2.2 | Chemical composition of the extract by
HPLC/UV-DAD
The analysis was performed using an HPLC apparatus (Waters, MA)
equipped with a UV/DAD detector. The method used is described by
(Gird et al., 2014). Briefly, stationary phase used is Nucleosil (C18, 25
3 0.4 mm, 5 mm particle). The mobile phase used is a mixture of two
solvents, the first is a mixture of water and phosphoric acid to 999:1
(v/v) (solvent A) and the second is acetonitrile (solvent B). The gradient
used was linear from 90 to 78% A and 10 to 22% B, 0–13 min; 78 to
60% A and 22 to 40% B, 13–14 min; 60% A and 40% B, 14–20 min.
Flow rate is 1.5 mL/min, injection volume is 20 ml, and detector wave-
length was fixed at 310 nm. The identification of separated compounds
was performed by comparing retention times of peaks in obtained
chromatogram with retention times of standards previously analyzed at
the same operating conditions, standards used are: caffeic acid (9.14
min), ferulic acid (12.39 min), rutin (15.59 min), rosmarinic acid (16.58
min), and quercitrin (18.87 min).
2.3 | Preparation and characterization of
microcapsules
2.3.1 | Preparation of microcapsules
Microcapsules were prepared by complex coacervation technique
according to methods previously described by (Mcmullen, Newton, &
Becker, 1982; Mcmullen, Newton, & Becker, 1984) with several modifi-
cations. Briefly, 0.5 g of the extract was mixed with 28.5 ml of gelatin
solution (5% w/v) at 40 8C (the protein denaturation temperature).
After 15 min of stirring, 11.5 ml of pectin solution (5% w/v) at
40 8C were added and the pH of reactional system was adjusted to 3
(the isoelectric point of the protein).
The system was cooled to 5 8C (cold gelification of micropar-
ticles), then 1 ml of formaldehyde was added and stirring is continued
for 30 min.
Finally, the pH of the system was adjusted to 9 using a sodium
hydroxide solution (20% w/v). After 2 hr of stirring, microcapsules
were washed using distilled water, filtered by filter paper Whatman
n81, and dried.
2.3.2 | Encapsulation yield
Encapsulations yields were determined using the following equation:
NAIT BACHIR ET AL.
Encapsulated amount of polyphenols ðgÞ
Encapsulation yield 5 3100
0:5
The amount of polyphenols introduced initially is known (repre-
sents 0.5 g) and the amount of encapsulated polyphenols was deter-
mined by assaying total polyphenols content in freeze-dried
microcapsules.
About 100 mg of microcapsules were finely crushed and mixed
with 100 ml of ethanol, after stirring for 1 hr the solution was filtered
using a syringe microfilter with 0.2 lm pore diameter and the amount
of total polyphenols was determined according to method previously
described by (Ainsworth & Gillespie, 2007; Folin & Ciocalteu, 1927).
The assay was carried out using a UV–Visible spectrophotometer
SHIMADZU (Double Monochromator UV-2700, Tokyo, Japan), 0.5 ml
of each solution was taken and mixed with 2.5 ml of sodium carbonate
solution (20% w/v), and 2.5 ml of Folin–Ciocalteu (10% v/v). After 2 hr
incubation at room temperature, the absorbance of solutions was
determined at 768 nm. The quantification was made according to a cal-
ibration curve established under the same conditions using Gallic acid
solutions with different concentrations (from 0 to 50 mg/mL). All meas-
urements were done in triplicate at 25 8C.
2.3.3 | Microscopic analysis
Microscopic observation of the obtained microcapsules was performed
at 403 magnification using an optical microscope (BX60 Olympus,
Tokyo, Japan).
2.3.4 | Determination of the mean diameter
The mean diameters of different microcapsules were determined by
image processing, the obtained microphotographs have been selected
and the mean diameters of microparticles are determined using Image-
J software (Version 1.6.0, MD).
2.4 | Preparation of suspensions
About 0.5 mg of each dispersing agent (xanthan gum, tragacanth, or
sodium alginate) was added to 89.5 ml of distilled water, after 2 hr of
stirring, polymer solutions were homogenized by Ultra-turrax (IKA, T25
digital, Staufen, Germany) at 10,000 rpm for 5 min. About 10 g of dried
microcapsules were introduced into each prepared solution and
obtained suspensions were homogenized again by ultra-turrax at
10,000 rpm for 5 min, in order to obtain a homogeneous dispersion of
microcapsules (dispersed phase) in polymer solution (continuous
phase). The formulations codes (S1: suspension stabilized by xanthan
gum, S2: suspension stabilized by tragacanth, and S3: suspension stabi-
lized by sodium alginate) and compositions are given in Table 1.
T A B LE 1 Formulations composition
Formulation code Microcapsules Xanthan gum
S1 10% 0.5%
S2 10% 2 0.5%
S3 10% 2 2
| 3 of 10
2.5 | Characterization of the prepared suspensions
2.5.1 | Mean diameters measurement
The average size of prepared suspensions was determined using
FRITSCH ANALYSETTE (22 MicroTec plus, Idar-Oberstein, Germany)
laser particle size. The mean diameter was calculated using the follow-
ing equation:
Pn
i51 fi :xi
DM 5 P n
i51 fi
where, DM is the arithmetic mean diameter, n is the number of classes
dividing the sample, xi is the representative diameter, and fi is the fre-
quency. All measurements were done in triplicate at 25 8C.
2.5.2 | Zeta potential measurement
Zeta potentials of the prepared suspensions were determined using
Nanotrac wave instrument (Microtrac Inc., PA), they have been calcu-
lated using Smoluchowski equation:
4ph v
f5
E UL
where, U is the voltage, L is the distance between the two electrodes, E
and h are the dielectric constant and the viscosity of pure water,
respectively, and m is the velocity of mobile microparticles in the elec-
tric field. All measurements were done in triplicate at 25 8C.
2.5.3 | Viscosity measurement
The suspensions viscosities were measured at 50 rpm using Brook
Field Viscometer apparatus (For LV-II viscosity range, MA). All meas-
urements were done in triplicate at 25 8C.
2.5.4 | Antioxidant activity evaluation
Antioxidant activity of the prepared suspension and of their different
constituents was determined using DPPH scavenging radical method,
according to the protocol described by (Molyneux, 2004; Quint~ao,
Tavares, Vieira-Filho, Souza, & Santos, 2013) with some modifications.
Briefly, DPPH solution at 0.004 g per 100 ml of methanol was prepared.
About 3 ml of the DPPH solution were mixed with 1 ml of solution to
be analyzed (5 mg/mL). After 30 min dark incubation, the absorbance of
each solution was measured at 517nm using SHIMADZU UV–visible
spectrophotometer (Double Monochromator UV-2700, Tokyo, Japan).
Antioxidant activity was determined using the following equation:
ðA0 2AS Þ
AAð%Þ5 3100
A0
where, AA is the percentage of the antioxidant activity, A0 is the absorb-
ance of the DPPH solution, and AS is the absorbance of the DPPH
Tragacanth Sodium alginate Purified water
2 2 89.5%
2 89.5%
0.5% 89.5%
4 of 10 | NAIT BACHIR
solution containing the sample to analyze. All measurements were done
in triplicate at 25 8C.
2.6 | Stability study of prepared suspensions
The suspensions were placed in cylindrical tubes of 20 cm height and
1.5 cm diameter, and then stored at 25 8C for 90 days. Every 30 days
tubes were recovered to estimate sedimentation volume and redisper-
sibility; after tube homogenization, a sample was taken to assess the
physicochemical and antioxidant properties of suspensions.
2.6.1 | Determination of physicochemical properties
Mean diameter, zeta potential, and viscosity were determined by the
same methods described above in Sections 2.5.1, 2.5.2, and 2.5.3,
respectively.
2.6.2 | Sedimentation volume
Sedimentation volume was calculated using the following equation
(Junyaprasert & Manwiwattanakul, 2008; Matthews & Rhodes, 1968):
Hs
Sedimentation volume5
H0
where, HS is the height of sediment and H0 is the initial height of
suspension.
2.6.3 | Redispersibility
The redispersibility was estimated by the number of 3608 turns at
20 RPM necessary for total homogenization of sedimented microcap-
sules (Junyaprasert & Manwiwattanakul, 2008; Matthews & Rhodes,
1968).
2.6.4 | Antioxidant activity degradation
Degradation of antioxidant activity for the various prepared suspen-
sions and for an extract solution at 5 mg/mL stored at 25 8C was
FIGURE 1 HPLC chromatogram of Salvia officinalis extract
ET AL.
carried out by estimating the antioxidant activity every 30 days for 90
days of storage using the method described in Section 2.5.4 above.
The modeling of antioxidant activity degradation kinetics was per-
formed using the first order model which is given by the following
equation:
dDAA
52kDAA
dt
where, DAA is the degradation of antioxidant activity during storage, t
is the storage time, and k is the constant of degradation.
After linearization of the equation, the constant k is determined
graphically; afterward the half-life time t1/2 is calculated using the fol-
lowing equation:
ln2
t1 5
2 k
3 | RESULTS AND DISCUSSION
3.1 | Preparation and chemical composition of Salvia
officinalis extract
Extraction yield is 27.11 6 3.20%. HPLC chromatogram of obtained
extract is given in Figure 1; the main extracted molecules are rutin
(44.88%), quercitrin (18.83%), rosmarinic acid (12.68%), caffeic acid
(6.36%), and ferulic acid (3.40%).
3.2 | Preparation and characterization of
microcapsules
The microencapsulation process of Salvia officinalis extract in pectin–
gelatin system allowed an encapsulation yield of 73.54 6 2.04%.
Figure 2 is a photomicrograph showing obtained microcapsules, image
processing showed the formation of spherical microcapsules with well-
NAIT BACHIR ET AL.
FIGURE 2 Photomicrograph showing the obtained microcapsules
defined edges, the average diameter of formed microcapsules is
51.12 6 2.86 mm.
3.3 | Preparation and characterization of suspensions
The prepared suspensions have similar organoleptic characteristics
(sage smell, green color, and homogeneous appearance). These organo-
leptic characteristics were determined by the evaluation of odor, color
and appearance of prepared suspensions by the sensory panel group
consisting of 1 man and 2 women.
3.3.1 | Physicochemical characteristics
Table 2 shows the physicochemical characteristics of three prepared
suspensions. The average diameters of suspensions S1, S2, and S3 are
52.80 6 3.05 mm, 54.10 6 4.58 mm, and 79.40 6 3.12 mm, respectively.
We note that microcapsules diameters increased after their suspension,
this increase is due to the adsorption of stabilizing agents on microcap-
sules surface. Effectively, xanthan gum, tragacanth, and sodium alginate
are anionic polymers which carry negative charges (George & Abraham,
2006) and gelatin–pectin based microcapsules present a positive sur-
face charge (Doublier, Garnier, Renard, & Sanchez, 2000), so the elec-
trostatic interactions allow adsorption of polymers on microcapsules
surface (Doublier et al. 2000; Fuoss & Strauss, 1948; Lankalapalli &
Kolapalli, 2009), hence the increase of their diameters.
Zeta potentials of suspensions S1, S2, and S3 are 217.23 6 0.99
mV, 213.41 6 0.77 mV, and 261.37 6 3.54 mV, respectively. Negative
values of zeta potential are due to negative charges on the different
T A B LE 2 Physicochemical characteristics of the prepared suspensions
Characteristics Suspension 1
Mean diameter (mm) 52.80 6 3.05
Zeta potential (mV) 217.23 6 20.99
Viscosity (Pa s) 31.52 6 1.82
* Statistically significant difference between suspension 1 and suspension 2 (p < .05).
( )
(
**)Nonsignificant difference between suspension 1 and suspension 2 (p > .05).
(
***)Statistically significant difference between suspension 1 and suspension 3 (p < .05).
(
****)Statistically highly significant difference between suspension 1 and suspension 3 (p < .001).
(#)
Statistically significant difference between suspension 2 and suspension 3 (p < .05).
(##)
Statistically highly significant difference between suspension 2 and suspension 3 (p < .001).
| 5 of 10
polyanions used in each formulation, the difference in zeta potentials
between prepared suspensions is due to the ability of each polymer to
adsorb on microparticle surface (Hajdu
s, Tomb
acz, & Borb


, Ille ath,
2009). It is noticed that zeta potential of suspension S3 is very high
compared with suspensions S1 and S2 which allows us to say that
sodium alginate adsorbs very efficiently on gelatin–pectin based micro-
capsules, this result allows explaining the important increase in mean
diameters of microcapsules content in S3.
Viscosities of suspensions S1, S2, and S3 are 31.52 6 1.82 Pa s,
27.05 6 1.56 Pa s, and 22.93 6 1.32 Pa s, respectively. The very high
viscosity of stabilized suspensions by xanthan gum and tragacanth (S1
and S2) compared with suspension S3 which is stabilized by sodium
alginate is explained by the fact that natural gums have a very high
thickening effect (Azarikia & Abbasi, 2010; Garcıa-Ochoa et al. 2000;
Saha & Bhattacharya, 2010) whereas the sodium alginate has a medium
thickening effect (Saha & Bhattacharya, 2010; Sugawara, Teruko, &
Otagiri, 1994).
3.3.2 | Antioxidant activity
Table 3 shows the values of antioxidant activity of prepared suspensions
and their various components. The antioxidant activities of free sage
extract, gelatin, pectin, prepared microcapsules, xanthan gum, tragacanth,
sodium alginate, S1, S2, and S3 are 97.49 6 4.11%, 48.97 6 3.45%,
47.11 6 2.88%, 96.89 6 5.81%, 42.84 6 1.85%, 47.83 6 2.35%, 34.69 6
2.24%, 96.46 6 5.57%, 95.29 6 5.50%, and 96.65 6 5.58%, respectively.
We note that antioxidant activity of sage extract is greater than
the activities of different polymers used for extract encapsulation (gela-
tin and pectin) and for stabilization of dispersions (xanthan gum, traga-
canth, and sodium alginate), this very strong antioxidant activity is due
to different polyphenols contained in the extract of Salvia officinalis.
Effectively, previously carried out HPLC analysis of sage extract
showed the dominance of flavonoids (rutin and quercitrin) and the
presence of phenolic acids (caffeic acid, ferulic acid, and rosmarinic
acid) (Gird et al., 2014; Walch, 2011; Walch, Tinzoh, Zimmermann,
Stuhlinger, & Lachenmeier, 2011).
We note that antioxidant activity of sage extract increases after
microencapsulation and dispersion, this result was predictable accord-
ing to studies carried out by (Donsì, Annunziata, Sessa, & Ferrari, 2011)
showing antimicrobial activity increase of D-limonene after its nanoe-
mulsification. Effectively, microscopic size which allows greater
Suspension 2 Suspension 3
54.10 6 3.12 **
( )
79.40 6 4.58(****) (##)
213.41 6 20.77(*) 261.37 6 23.54(****) (##)
27.05 6 1.56(*) 22.93 6 1.32(***) (#)
6 of 10 | NAIT BACHIR ET AL.

T A B LE 3 Antioxidant activity of formulated suspensions and their

different constituents

Systemsa Contentsb AAc (%)

Sage extract (5 mg/mL) 5 mg 97.49 6 4.11

Gelatin (5 mg/mL) 5 mg 48.97 6 3.45

Pectin (5 mg/mL) 5 mg 47.11 6 2.88

Microcapsules (5 mg mL) 1.250 mg of Sage extract. 96.89 6 5.81

2.680 mg of gelatin.
1.070 mg of pectin.

Xanthan gum (5 mg/mL) 5mg 42.84 6 1.85

Tragacanth (5 mg/mL) 5mg 47.83 6 2.35

Sodium alginate (5 mg mL) 5mg 34.69 6 2.24

S1 (5 mg/mL) 0.125 mg of Sage extract. 96.46 6 5.57

0.268 mg of gelatin. FIGURE 4 Zeta potential evolution of the suspensions during
0.107 mg of pectin. storage
0.025 mg of xanthan gum.

S2 (5 mg/mL) 0.125 mg of Sage extract. 95.29 6 5.50 3.5 | Effect of storage time on the physicochemical
0.268 mg of gelatin. characteristics
0.107 mg of pectin.
0.025 mg of taraghant gum. Figure 3 shows the evolution of microcapsules mean diameters during
S3 (5 mg/mL) 0.125 mg of Sage extract. 96.65 6 5.58 storage for different formulations. Mean diameters of suspensions S1
0.268 mg of gelatin. and S2 remain almost stable during storage. The mean diameters of
0.107 mg of pectin. microcapsules content in suspension S3 increase after 30 days of stor-
0.025 mg of sodium alginate.
age, this increase is due to the continuity of sodium alginate fixation on
a
Systems represent different formulations or raw materials used in the microcapsules surfaces; Beyond 30 days of storage, we notice that
formulations.
b
mean diameters become more stable. Effectively, adsorption time of a
The chemical composition of each system (5 mg).
c
The percentage of the antioxidant activity. polymer on a surface may take several days before entering into the
equilibrium phase (Frantz & Granick, 1991).
interaction of actives with specific reagents, cells or tissues, rough sur- Figure 4 shows the evolution of zeta potential suspensions during
face of microparticles provides better interactions with reagents and storage. Zeta potentials of suspensions S1 and S2 remain practically
biological systems. stable during storage, while suspension S3 has a slight elevation of zeta
potential after 30 days of storage; this elevation was due to the
3.4 | Stability study of the suspensions increase of macromolecular cloud around microcapsules which is prin-

During storage, there was no change in organoleptic characteristics of cipally composed of sodium alginate chains negatively charged.

prepared suspensions (color, odor, and appearance).

FIGURE 3 Evolution of microcapsules means diameters during storage FIGURE 5 Viscosity evolution of the suspensions during storage
NAIT BACHIR ET AL.
FIGURE 6 Suspensions photographs showing the evolution of sedimentation phenomenon
Figure 5 shows the variation of suspensions viscosities during stor-
age. Viscosities of suspensions S1 and S2 remain practically stable dur-
ing storage while suspension S3 exhibits a slight increase in viscosity
after 30 days of storage; this is due to the increase in the mean diame-
ter of microcapsules contained in various suspensions. Effectively,
when the particle size of dispersion increases its viscosity also
increases (Maranzano & Wagner, 2001).
3.5.1 | Effect of storage time on the microcapsules
sedimentation
Figure 6 presents photographs of suspensions after formulation, after
30 days, after 60 days and after 90 days of storage at 25 8C, clearly
showing the evolution of the sedimentation phenomenon.
Figure 7 shows the evolution of sedimentation volumes during
storage for each suspension. After 30 days of storage the sedimenta-
tion volume of suspension S3 is lower than suspensions S1 and S2, this
result is due to the viscosity difference. Effectively, when the viscosity
of dispersion increases the sedimentation rate of dispersed particles
decreases (Tsubaki, Kato, Miyazawa, Kuma, & Mori, 2001). After 90
days of storage, suspensions S1 and S2 have a very low sedimentation
FIGURE 7 Evolution of the suspensions sedimentation volumes
during storage
| 7 of 10
volume compared with suspension S3; this is due to the important
diameter of microcapsules forming the suspension S3 and the very
large repulsion forces between various microcapsules because of their
very important zeta potential values.
3.5.2 | Study of the suspensions redispersibility and
proposition of the stabilization mechanisms
Figure 8 shows the evolution of suspensions redispersibility during
storage, the results show that when the storage time increases, the
redispersibility becomes more difficult.
Suspension S3 has a faster redispersibility compared with sus-
pensions S1 and S2, because its sedimentation volume is more
important.
The proposed mechanism for the stabilization of suspension S3 by
sodium alginate is shown in Figure 9; it is based on the ability of these
negatively charged macromolecules to adsorb on the microcapsules
surface, which increases their diameters and their zeta potential. Hence
the increase in both repulsion forces between particles, and spaces
between the sedimented particles.
FIGURE 8 Evolution of the suspensions redispersibility during
storage
8 of 10 | NAIT BACHIR ET AL.

FIGURE 9 Stabilization mechanisms proposed for each dispersant agent

Suspension S2 has a faster Redispersibility compared with suspen- low antioxidant activity degradation comparing to free extract of Salvia
sion S1. The proposed mechanism of suspension S2 stabilization by officinalis.
tragacanth is shown in Figure 9, it is based on the chemical composi- Values of the first order kinetic constants “K,” correlation coeffi-
tion of tragacanth. cients “R2,” and half-life time “t1/2” are given in Table 4. The half-life
Tragacanth is composed of two polysaccharides: tragacanthine is a times of S1, S2, S3 and free sage extract are 258.64 6 21.99 days
water soluble hydrocolloid which increases suspension viscosity (R2 5 .98), 230.28 6 21.92 days (R2 5 .97), 245.80 6 32.17 days
(Azarikia & Abbasi, 2010; Balaghi, Mohammadifar, Zargaraan, Gavlighi, (R2 5 .95), and 12.75 6 1.95 days (R2 5 .93), respectively.
& Mohammadi, 2011) and water insoluble bassorins which sediment at We observe that their values for the three suspensions are very
the same time as microcapsules (Azarikia & Abbasi, 2010). The sedi- similar, while for the free extract the value of kinetic constant “K”
mented bassorins allow mechanical separation between different sedi-
mented microcapsules, this avoids the flocculation and aggregation
phenomena of sedimented microcapsules and can also ease their
redispersibility.
The suspension S1 stabilized with xanthan gum represents the
most difficult redispersibility, this is due to the stabilization mechanism
of suspension S1 by xanthan gum which is based principally on the vis-
cosifying power of polymer (Figure 9) which allows to slow down the
sedimentation, but once the microcapsules are sedimented they begin
to form aggregates (Rawlins & Kayes, 1983) and their redispersibility
becomes very difficult.

3.5.3 | Effect of storage time on the antioxidant activity

degradation
Figure 10 shows the evolution of antioxidant activity degradation of FIGURE 10 Evolution of the antioxidant activity degradation of
suspensions and free sage extract during storage. Suspensions have a suspensions (S1, S2, and S3) and free extract during storage
NAIT BACHIR ET AL.
T A B LE 4 The values of the kinetic constants K and the half-life
time t1/2 for the suspensions and free extract
K 3 1023 (Days21) R2 t1/2 (Days)
Suspension S1 2.68 6 0.23 .98 258.64 6 21.99
Suspension S2 3.01 6 0.29 .97 230.28 6 21.92
Suspension S3 2.82 6 0.37 .95 245.80 6 32.17
Free Sage extract 54.37 6 8.30 .93 12.75 6 1.95
increases and the value of half-life time “t1/2” decreases in a very
important way.
The decrease in antioxidant activity of sage extract is due to the
oxidation of polyphenols by water and oxygen, and these reactions are
catalyzed by environmental conditions such as light and temperature
(Fang & Bhandari, 2010; Shahidi & Han, 1993). Suspensions containing
the encapsulated extract in a gelatin–pectin based membrane sur-
rounded by stabilizers (xanthan gum, tragacanth, or sodium alginate)
exert a protective effect on bioactive molecules contained in sage
extract against the environmental assaults.
4 | CONCLUSIONS
Salvia officinalis extract is mainly composed of polyphenols (caffeic acid,
ferulic acid, rosmarinic acid, rutin, and quercetin), it can be encapsu-
lated in a gelatin–pectin membrane using the complex coacervation
method.
This work allowed to propose the stabilization mechanisms based
microcapsules suspensions prepared using xanthan gum, tragacanth,
and sodium alginate as a dispersing agent. The large number of nega-
tive charges on the polymeric chains of sodium alginate and its high
capacity to adsorb on the positively charged surfaces, permit it to pro-
vide the stabilization of the dispersion for 90 days at 258 C.
Antioxidant activity of sage extract increases after its formulation;
furthermore, degradation of this activity during storage decreased
because of the protective effect of polymers used on bioactive mole-
cules composing the sage extract.
This work can be the basis for the development of commercial prod-
ucts containing plant extracts in order to delay their expiration dates.
ORCID
Yacine Nait Bachir http://orcid.org/0000-0002-6361-7935
Amel Zafour http://orcid.org/0000-0002-9625-2602
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