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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
Abstract
Technology, University of Saad Dahlab-Blida The stabilization of microparticles suspensions is always assured by macromolecules that allow
1, Blida, Algeria the increasing of dispersing phase viscosity and maintaining the microparticles dispersibility. In
2
Laboratory of Natural Bio-Resources, this work, the effect of xanthan gum, tragacanth, and sodium alginate on the stability of gelatin–
Department of Biology, Faculty of Science,
pectin microcapsules suspensions containing sage polyphenols as an active substance was inves-
Hassiba Benbouali University of Chlef, Chlef,
Algeria tigated. After polyphenols extraction from Salvia officinalis, their characterization by HPLC-UV/
DAD and their microencapsulation by complex coacervation technique using gelatin and pectin.
Correspondence Three different suspensions of microcapsules were prepared using different polymeric dispers-
Yacine Nait Bachir, Chemical Engineering
ant agents. The suspensions were characterized by laser for particle size, zetametry,
Laboratory, Process Engineering
Department, Faculty of Technology, viscosimetry, and evaluation of their antioxidant activities was carried out by the DPPH scav-
University of Saad Dahlab-Blida 1, Algeria. enging radical method. Stability study of prepared suspensions was undertaken during 90 days,
Email: phd.nait.bachir.yacine@gmail.com
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 of this phase in continuous phase (Vincent, 1974). On the other hand,
the chemical instability is mainly due to the effect of water compos-
The stabilization of suspensions containing microparticles is a purpose ing the dispersing phase on different dispersed ingredients (Di Mattia,
that different industries look to achieve. Effectively, several active Sacchetti, Mastrocola, & Pittia, 2009).
ingredients used in different industries are insoluble in aqueous solu- Xanthan gum is a natural polysaccharide of microbial origin that
tions and present a very low physicochemical stability in these environ- presents high viscosifying effect (Garcıa-Ochoa, Santos, Casas, &
ments, as some drugs (Blessy, Patel, Prajapati, & Agrawal, 2014), Gomez, 2000). Tragacanth is a natural gum composed of two differ-
preservatives (De Vos, Faas, Spasojevic, & Sikkema, 2010), antioxidants ent types of polysaccharides, tragacanthine (30–40%) which is a
(Hernandez-Jaimes, Fouconnier, Pe
rez-Alonso, Munguía-Guille
n, & water-soluble hydrocolloid and bassorin (60–70%) which is insoluble
Vernon-Carter, 2013), pesticides (Szente, 1998), bacteria (Muhammad, in water and swells for forming a hydrogel (Azarikia & Abbasi, 2010).
Ramzan, Huo, Tian, & Bian, 2017), and chemical catalysts (Scott, Datye, Alginates are natural polysaccharides extracted from marine algae
& Crooks, 2003), used by pharmaceutical, food, cosmetics, phytosani- having a high negative charge (George & Abraham, 2006). The com-
tary, and chemical industries, respectively. mon point of these three different polymers previously described
Physical instability of these suspensions is described by a rapid (xanthan gum, tragacanth, and sodium alginate) is their ability to
separation of the dispersed phase and a very difficult redispersibility increase the viscosity of an aqueous solutions (Azarikia & Abbasi,

J Food Process Preserv. 2017;e13446. wileyonlinelibrary.com/journal/jfpp V


C 2017 Wiley Periodicals, Inc. | 1 of 10
https://doi.org/10.1111/jfpp.13446
2 of 10 | NAIT BACHIR ET AL.

2010; Garcıa-Ochoa et al., 2000; George & Abraham, 2006; Taher- 2.2 | Preparation and characterization of sage extract
ian, Fustier, Britten, & Ramaswamy, 2008), which allows them to be
2.2.1 | Preparation of extract
used as dispersing agents.
Salvia officinalis is a plant of Mediterranean origin and member of The extraction was performed by a Soxhlet extractor (Riviera, 500 ml
the mint family Lamiaceae, it is traditionally used in cooking, infusion capacity, Mumbai, India) using ethanol as extraction solvent. About
preparation, and aromatherapy. 50 g of plant powder was introduced into a cellulose extraction thim-
This aromatic plant containing essential oil and polyphenols is one ble, the thimble was placed in a Soxhlet, equipped with a 1 L distillation
of the most widely plants used in cosmetics (Silvestin et al., 2012), flask containing 750 ml of ethanol. Heating was provided with a heat-
pharmaceutical (Hamidpour, Hamidpour, Hamidpour, & Shahlari, 2014), ing mantle (Electrothermal, Staffordshire, UK). After 120 min of extrac-
and food industries (Hayouni et al., 2008). tion, the extract was recovered after solvent evaporation.
The problem encountered with these bioactive molecules
extracted from sage and other plants is their great sensitivity to envi- 2.2.2 | Chemical composition of the extract by
ronmental conditions like light, temperature, humidity, and oxygen HPLC/UV-DAD
(Desai & Jin Park, 2005). Effectively, according to a study reported by The analysis was performed using an HPLC apparatus (Waters, MA)
€hlinger, & Monakhova, 2012) the pol-
(Walch, Lachenmeier, Kuballa, Stu equipped with a UV/DAD detector. The method used is described by
yphenols of sage infusion are completely reduced after 60 hr of stor- (Gird et al., 2014). Briefly, stationary phase used is Nucleosil (C18, 25
age, this is why we have chosen this plant as a model to study the 3 0.4 mm, 5 mm particle). The mobile phase used is a mixture of two
preservation of the bioactive molecules extracted from plants. solvents, the first is a mixture of water and phosphoric acid to 999:1
The originality of the present work is to study the effect of differ- (v/v) (solvent A) and the second is acetonitrile (solvent B). The gradient
ent polymeric dispersing agents on the physicochemical stability of an used was linear from 90 to 78% A and 10 to 22% B, 0–13 min; 78 to
active ingredient which has been previously encapsulated in a poly- 60% A and 22 to 40% B, 13–14 min; 60% A and 40% B, 14–20 min.
meric membrane. At first, bioactive molecules are extracted from Salvia Flow rate is 1.5 mL/min, injection volume is 20 ml, and detector wave-
officinalis, characterized by HPLC-UV/DAD, and then encapsulated by length was fixed at 310 nm. The identification of separated compounds
complex coacervation technique using gelatin (polycation) and pectin was performed by comparing retention times of peaks in obtained
(polyanion). After that, the suspensions based on these microcapsules chromatogram with retention times of standards previously analyzed at
are prepared using different dispersing agents (xanthan gum, traga- the same operating conditions, standards used are: caffeic acid (9.14
canth, and sodium alginate), the suspensions will be characterized by min), ferulic acid (12.39 min), rutin (15.59 min), rosmarinic acid (16.58
laser particle size, zetametry, viscosimetry, and their antioxidant activ- min), and quercitrin (18.87 min).
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- 2.3 | Preparation and characterization of
bilization mechanisms will be proposed for each dispersing agent. microcapsules
2.3.1 | Preparation of microcapsules
2 | MATERIALS AND METHODS Microcapsules were prepared by complex coacervation technique
according to methods previously described by (Mcmullen, Newton, &
2.1 | Materials 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
Xanthan gum (Rhodia, Algiers, Algeria), Gelatin type B from bovine skin
solution (5% w/v) at 40 8C (the protein denaturation temperature).
(Sigma Aldrich, Taufkirchen, Germany), and sodium alginate (Sigma
After 15 min of stirring, 11.5 ml of pectin solution (5% w/v) at
Aldrich, Deisenhofen, Germany) were provided by the pharmaceutical
40 8C were added and the pH of reactional system was adjusted to 3
group Saidal (Algiers, Algeria). Tragacanth fine powder (Merck, Darm-
(the isoelectric point of the protein).
stadt, Germany), pectin from Citrus fruits (Sigma Aldrich, Taufkirchen,
The system was cooled to 5 8C (cold gelification of micropar-
Germany), Folin–Ciocalteu (Sigma Aldrich, Taufkirchen, Germany),
ticles), then 1 ml of formaldehyde was added and stirring is continued
diphenyl-picryl-hydrazyl DPPH (Sigma Aldrich, Taufkirchen, Germany),
for 30 min.
and gallic acid 1-hydrate (Panreac quimica, Barcelona, Spain) were
Finally, the pH of the system was adjusted to 9 using a sodium
purchased.
hydroxide solution (20% w/v). After 2 hr of stirring, microcapsules
HPLC standards: caffeic acid, ferulic acid, rutin, rosmarinic acid,
were washed using distilled water, filtered by filter paper Whatman
and quercitrin were purchased from Sigma Aldrich (St. Louis, MO,
n81, and dried.
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 2.3.2 | Encapsulation yield
chemical products and distilled water were of analytical grade. Encapsulations yields were determined using the following equation:
NAIT BACHIR ET AL. | 3 of 10

Encapsulated amount of polyphenols ðgÞ 2.5 | Characterization of the prepared suspensions


Encapsulation yield 5 3100
0:5
2.5.1 | Mean diameters measurement
The amount of polyphenols introduced initially is known (repre-
The average size of prepared suspensions was determined using
sents 0.5 g) and the amount of encapsulated polyphenols was deter-
FRITSCH ANALYSETTE (22 MicroTec plus, Idar-Oberstein, Germany)
mined by assaying total polyphenols content in freeze-dried
laser particle size. The mean diameter was calculated using the follow-
microcapsules.
About 100 mg of microcapsules were finely crushed and mixed ing equation:
Pn
i51 fi :xi
with 100 ml of ethanol, after stirring for 1 hr the solution was filtered
DM 5 P n
using a syringe microfilter with 0.2 lm pore diameter and the amount i51 fi

of total polyphenols was determined according to method previously where, DM is the arithmetic mean diameter, n is the number of classes
described by (Ainsworth & Gillespie, 2007; Folin & Ciocalteu, 1927). dividing the sample, xi is the representative diameter, and fi is the fre-
The assay was carried out using a UV–Visible spectrophotometer quency. All measurements were done in triplicate at 25 8C.
SHIMADZU (Double Monochromator UV-2700, Tokyo, Japan), 0.5 ml
of each solution was taken and mixed with 2.5 ml of sodium carbonate 2.5.2 | Zeta potential measurement
solution (20% w/v), and 2.5 ml of Folin–Ciocalteu (10% v/v). After 2 hr Zeta potentials of the prepared suspensions were determined using
incubation at room temperature, the absorbance of solutions was Nanotrac wave instrument (Microtrac Inc., PA), they have been calcu-
determined at 768 nm. The quantification was made according to a cal- lated using Smoluchowski equation:
ibration curve established under the same conditions using Gallic acid
4ph v
solutions with different concentrations (from 0 to 50 mg/mL). All meas- f5
E UL
urements were done in triplicate at 25 8C.
where, U is the voltage, L is the distance between the two electrodes, E
2.3.3 | Microscopic analysis and h are the dielectric constant and the viscosity of pure water,
respectively, and m is the velocity of mobile microparticles in the elec-
Microscopic observation of the obtained microcapsules was performed
tric field. All measurements were done in triplicate at 25 8C.
at 403 magnification using an optical microscope (BX60 Olympus,
Tokyo, Japan). 2.5.3 | Viscosity measurement
The suspensions viscosities were measured at 50 rpm using Brook
2.3.4 | Determination of the mean diameter
Field Viscometer apparatus (For LV-II viscosity range, MA). All meas-
The mean diameters of different microcapsules were determined by
urements were done in triplicate at 25 8C.
image processing, the obtained microphotographs have been selected
and the mean diameters of microparticles are determined using Image- 2.5.4 | Antioxidant activity evaluation
J software (Version 1.6.0, MD). Antioxidant activity of the prepared suspension and of their different
constituents was determined using DPPH scavenging radical method,
2.4 | Preparation of suspensions according to the protocol described by (Molyneux, 2004; Quint~ao,
Tavares, Vieira-Filho, Souza, & Santos, 2013) with some modifications.
About 0.5 mg of each dispersing agent (xanthan gum, tragacanth, or
Briefly, DPPH solution at 0.004 g per 100 ml of methanol was prepared.
sodium alginate) was added to 89.5 ml of distilled water, after 2 hr of
About 3 ml of the DPPH solution were mixed with 1 ml of solution to
stirring, polymer solutions were homogenized by Ultra-turrax (IKA, T25
be analyzed (5 mg/mL). After 30 min dark incubation, the absorbance of
digital, Staufen, Germany) at 10,000 rpm for 5 min. About 10 g of dried
each solution was measured at 517nm using SHIMADZU UV–visible
microcapsules were introduced into each prepared solution and
spectrophotometer (Double Monochromator UV-2700, Tokyo, Japan).
obtained suspensions were homogenized again by ultra-turrax at
Antioxidant activity was determined using the following equation:
10,000 rpm for 5 min, in order to obtain a homogeneous dispersion of
microcapsules (dispersed phase) in polymer solution (continuous ðA0 2AS Þ
AAð%Þ5 3100
phase). The formulations codes (S1: suspension stabilized by xanthan A0

gum, S2: suspension stabilized by tragacanth, and S3: suspension stabi- where, AA is the percentage of the antioxidant activity, A0 is the absorb-
lized by sodium alginate) and compositions are given in Table 1. ance of the DPPH solution, and AS is the absorbance of the DPPH

T A B LE 1 Formulations composition

Formulation code Microcapsules Xanthan gum Tragacanth Sodium alginate Purified water

S1 10% 0.5% 2 2 89.5%

S2 10% 2 0.5% 2 89.5%

S3 10% 2 2 0.5% 89.5%


4 of 10 | NAIT BACHIR ET AL.

solution containing the sample to analyze. All measurements were done carried out by estimating the antioxidant activity every 30 days for 90
in triplicate at 25 8C. days of storage using the method described in Section 2.5.4 above.
The modeling of antioxidant activity degradation kinetics was per-
2.6 | Stability study of prepared suspensions formed using the first order model which is given by the following
equation:
The suspensions were placed in cylindrical tubes of 20 cm height and
dDAA
1.5 cm diameter, and then stored at 25 8C for 90 days. Every 30 days 52kDAA
dt
tubes were recovered to estimate sedimentation volume and redisper-
sibility; after tube homogenization, a sample was taken to assess the where, DAA is the degradation of antioxidant activity during storage, t

physicochemical and antioxidant properties of suspensions. is the storage time, and k is the constant of degradation.
After linearization of the equation, the constant k is determined
2.6.1 | Determination of physicochemical properties graphically; afterward the half-life time t1/2 is calculated using the fol-
Mean diameter, zeta potential, and viscosity were determined by the lowing equation:
same methods described above in Sections 2.5.1, 2.5.2, and 2.5.3, ln2
t1 5
respectively. 2 k

2.6.2 | Sedimentation volume


Sedimentation volume was calculated using the following equation 3 | RESULTS AND DISCUSSION
(Junyaprasert & Manwiwattanakul, 2008; Matthews & Rhodes, 1968):
3.1 | Preparation and chemical composition of Salvia
Hs
Sedimentation volume5
H0
officinalis extract

where, HS is the height of sediment and H0 is the initial height of Extraction yield is 27.11 6 3.20%. HPLC chromatogram of obtained
suspension. extract is given in Figure 1; the main extracted molecules are rutin
(44.88%), quercitrin (18.83%), rosmarinic acid (12.68%), caffeic acid
2.6.3 | Redispersibility (6.36%), and ferulic acid (3.40%).
The redispersibility was estimated by the number of 3608 turns at
20 RPM necessary for total homogenization of sedimented microcap-
3.2 | Preparation and characterization of
sules (Junyaprasert & Manwiwattanakul, 2008; Matthews & Rhodes,
microcapsules
1968).
The microencapsulation process of Salvia officinalis extract in pectin–
2.6.4 | Antioxidant activity degradation gelatin system allowed an encapsulation yield of 73.54 6 2.04%.
Degradation of antioxidant activity for the various prepared suspen- Figure 2 is a photomicrograph showing obtained microcapsules, image
sions and for an extract solution at 5 mg/mL stored at 25 8C was processing showed the formation of spherical microcapsules with well-

FIGURE 1 HPLC chromatogram of Salvia officinalis extract


NAIT BACHIR ET AL. | 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, Tombacz, & 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
FIGURE 2 Photomicrograph showing the obtained microcapsules
alginate is explained by the fact that natural gums have a very high
defined edges, the average diameter of formed microcapsules is thickening effect (Azarikia & Abbasi, 2010; Garcıa-Ochoa et al. 2000;
51.12 6 2.86 mm. Saha & Bhattacharya, 2010) whereas the sodium alginate has a medium
thickening effect (Saha & Bhattacharya, 2010; Sugawara, Teruko, &
Otagiri, 1994).
3.3 | Preparation and characterization of suspensions
The prepared suspensions have similar organoleptic characteristics 3.3.2 | Antioxidant activity
(sage smell, green color, and homogeneous appearance). These organo- Table 3 shows the values of antioxidant activity of prepared suspensions
leptic characteristics were determined by the evaluation of odor, color and their various components. The antioxidant activities of free sage
and appearance of prepared suspensions by the sensory panel group extract, gelatin, pectin, prepared microcapsules, xanthan gum, tragacanth,
consisting of 1 man and 2 women. 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
3.3.1 | Physicochemical characteristics 2.24%, 96.46 6 5.57%, 95.29 6 5.50%, and 96.65 6 5.58%, respectively.
Table 2 shows the physicochemical characteristics of three prepared We note that antioxidant activity of sage extract is greater than
suspensions. The average diameters of suspensions S1, S2, and S3 are the activities of different polymers used for extract encapsulation (gela-
52.80 6 3.05 mm, 54.10 6 4.58 mm, and 79.40 6 3.12 mm, respectively. tin and pectin) and for stabilization of dispersions (xanthan gum, traga-
We note that microcapsules diameters increased after their suspension, canth, and sodium alginate), this very strong antioxidant activity is due
this increase is due to the adsorption of stabilizing agents on microcap- to different polyphenols contained in the extract of Salvia officinalis.
sules surface. Effectively, xanthan gum, tragacanth, and sodium alginate Effectively, previously carried out HPLC analysis of sage extract
are anionic polymers which carry negative charges (George & Abraham, showed the dominance of flavonoids (rutin and quercitrin) and the
2006) and gelatin–pectin based microcapsules present a positive sur- presence of phenolic acids (caffeic acid, ferulic acid, and rosmarinic
face charge (Doublier, Garnier, Renard, & Sanchez, 2000), so the elec- acid) (Gird et al., 2014; Walch, 2011; Walch, Tinzoh, Zimmermann,
trostatic interactions allow adsorption of polymers on microcapsules Stuhlinger, & Lachenmeier, 2011).
surface (Doublier et al. 2000; Fuoss & Strauss, 1948; Lankalapalli & We note that antioxidant activity of sage extract increases after
Kolapalli, 2009), hence the increase of their diameters. microencapsulation and dispersion, this result was predictable accord-
Zeta potentials of suspensions S1, S2, and S3 are 217.23 6 0.99 ing to studies carried out by (Donsì, Annunziata, Sessa, & Ferrari, 2011)
mV, 213.41 6 0.77 mV, and 261.37 6 3.54 mV, respectively. Negative showing antimicrobial activity increase of D-limonene after its nanoe-
values of zeta potential are due to negative charges on the different mulsification. Effectively, microscopic size which allows greater

T A B LE 2 Physicochemical characteristics of the prepared suspensions

Characteristics Suspension 1 Suspension 2 Suspension 3

Mean diameter (mm) 52.80 6 3.05 54.10 6 3.12 **


( )
79.40 6 4.58(****) (##)

Zeta potential (mV) 217.23 6 20.99 213.41 6 20.77(*) 261.37 6 23.54(****) (##)

Viscosity (Pa s) 31.52 6 1.82 27.05 6 1.56(*) 22.93 6 1.32(***) (#)

* 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).
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. | 7 of 10

FIGURE 6 Suspensions photographs showing the evolution of sedimentation phenomenon

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

FIGURE 7 Evolution of the suspensions sedimentation volumes FIGURE 8 Evolution of the suspensions redispersibility during
during storage 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. | 9 of 10

T A B LE 4 The values of the kinetic constants K and the half-life characterization of gum tragacanth exudates from six species of Ira-
time t1/2 for the suspensions and free extract nian Astragalus. Food Hydrocolloids, 25(7), 1775–1784.
Blessy, M., Patel, R. D., Prajapati, P. N., & Agrawal, Y. K. (2014). Devel-
K 3 1023 (Days21) R2 t1/2 (Days)
opment of forced degradation and stability indicating studies of
Suspension S1 2.68 6 0.23 .98 258.64 6 21.99 drugs—A review. Journal of Pharmaceutical Analysis, 4(3), 159–165.

Suspension S2 3.01 6 0.29 .97 230.28 6 21.92 De Vos, P., Faas, M. M., Spasojevic, M., & Sikkema, J. (2010). Encapsula-
tion for preservation of functionality and targeted delivery of bioac-
Suspension S3 2.82 6 0.37 .95 245.80 6 32.17 tive food components. International Dairy Journal, 20(4), 292–302.
Free Sage extract 54.37 6 8.30 .93 12.75 6 1.95 Desai, K. G. H., & Jin Park, H. (2005). Recent developments in
microencapsulation of food ingredients. Drying Technology, 23(7),
1361–1394.
increases and the value of half-life time “t1/2” decreases in a very
Di Mattia, C. D., Sacchetti, G., Mastrocola, D., & Pittia, P. (2009). Effect
important way. of phenolic antioxidants on the dispersion state and chemical stabil-
The decrease in antioxidant activity of sage extract is due to the ity of olive oil O/W emulsions. Food Research International, 42(8),
oxidation of polyphenols by water and oxygen, and these reactions are 1163–1170.

catalyzed by environmental conditions such as light and temperature Donsì, F., Annunziata, M., Sessa, M., & Ferrari, G. (2011). Nanoencapsula-
tion of essential oils to enhance their antimicrobial activity in foods.
(Fang & Bhandari, 2010; Shahidi & Han, 1993). Suspensions containing
LWT - Food Science and Technology, 44(9), 1908–1914.
the encapsulated extract in a gelatin–pectin based membrane sur-
Doublier, J. L., Garnier, C., Renard, D., & Sanchez, C. (2000). Protein-
rounded by stabilizers (xanthan gum, tragacanth, or sodium alginate) polysaccharide interactions. Current Opinion in Colloid and Interface
exert a protective effect on bioactive molecules contained in sage Science, 5(3–4), 202–214.
extract against the environmental assaults. Fang, Z., & Bhandari, B. (2010). Encapsulation of polyphenols - A review.
Trends in Food Science and Technology, 21(10), 510–523.
Folin, O., & Ciocalteu, V. (1927). Tyrosine and tryptophan in proteins.
4 | CONCLUSIONS
Journal of Biological Chemistry, 73(2), 627–648.
Frantz, P., & Granick, S. (1991). Kinetics of polymer adsorption and
Salvia officinalis extract is mainly composed of polyphenols (caffeic acid,
desorption. Physical Review Letters, 66(7), 899–902.
ferulic acid, rosmarinic acid, rutin, and quercetin), it can be encapsu-
Fuoss, R. M., & Strauss, U. P. (1948). Electrostatic interaction of polye-
lated in a gelatin–pectin membrane using the complex coacervation lectrolytes and simple electrolytes. Journal of Polymer Science, 3(4),
method. 602–603.
This work allowed to propose the stabilization mechanisms based Garcıa-Ochoa, F., Santos, V. E., Casas, J. A., & Gomez, E. (2000). Xanthan
microcapsules suspensions prepared using xanthan gum, tragacanth, gum: Production, recovery, and properties. Biotechnology Advances,
18(1), 54–579.
and sodium alginate as a dispersing agent. The large number of nega-
George, M., & Abraham, T. E. (2006). Polyionic hydrocolloids for the
tive charges on the polymeric chains of sodium alginate and its high
intestinal delivery of protein drugs: Alginate and chitosan - A review.
capacity to adsorb on the positively charged surfaces, permit it to pro- Journal of Controlled Release, 114(1), 1–14.
vide the stabilization of the dispersion for 90 days at 258 C. Gird, C. E., Nencu, I., Costea, T., Duţu, L. E., Popescu, M. L., & Ciupitu,
Antioxidant activity of sage extract increases after its formulation; N. (2014). Quantitative analysis of phenolic compounds from salvia
furthermore, degradation of this activity during storage decreased officinalis l. leaves. Farmacia, 62(4), 649–657.

because of the protective effect of polymers used on bioactive mole- Hajdu s, E., Tombacz, E., & Borbath, I. (2009). Surface charging,
, A., Ille
polyanionic coating and colloid stability of magnetite nanoparticles.
cules composing the sage extract.
Colloids and Surfaces A: Physicochemical and Engineering Aspects,
This work can be the basis for the development of commercial prod- 347(1–3), 104–108.
ucts containing plant extracts in order to delay their expiration dates. Hamidpour, M., Hamidpour, R., Hamidpour, S., & Shahlari, M. (2014).
Chemistry, pharmacology, and medicinal property of sage (salvia) to
prevent and cure illnesses such as obesity, diabetes, depression,
ORCID
dementia, lupus, autism, heart disease, and cancer. Journal of Tradi-
Yacine Nait Bachir http://orcid.org/0000-0002-6361-7935 tional and Complementary Medicine, 4(2), 82–88.
Amel Zafour http://orcid.org/0000-0002-9625-2602 Hayouni, E. A., Chraief, I., Abedrabba, M., Bouix, M., Leveau, J.,
Mohammed, H., & Hamdi, M. (2008). Tunisian Salvia officinalis L. and
Schinus molle L. essential oils: Their chemical compositions and their
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