Youngjae Byun, Jin Bong Hwang, Sung Hwan Bang

Formulation and characterization of a-tocopherol loaded poly 3-caprolactone
(PCL) nanoparticles
Youngjae Byun a , Jin Bong Hwang c , Sung Hwan Bang d , Duncan Darby a , Kay Cooksey a , Paul L. Dawson b ,
Hyun Jin Park a,d , Scott Whiteside a, *
a Department of Packaging Science, B-212 Poole & Agricultural Center, Clemson University, Clemson, SC 29634-0320, USA
b Department of Food Science, 204 Poole & Agricultural Center, Clemson University, Clemson, SC 29634-0320, USA
c Korea Food Research Institute, 516 Bakhyun-dong, Bundang-gu, Sungnam-Si, Gyeonggi-do, 463-746, Republic of Korea
d Department of Food Technology, 306 School of Life Science and Biotechnology, Korea University, Seoul, 136-701, Republic of Korea
article info
Article history:
Received 27 January 2010
Received in revised form
24 June 2010
Accepted 27 June 2010
Keywords:
Polycaprolactone
Antioxidant
Emulsion
Ultrasonification
a-tocopherol
1. Introduction
abstract
Free radicals or, more generally, reactive oxygen species
(ROS) are products of normal cellular metabolism. They are the
molecules or molecular fragments containing unpaired electrons.
The unpaired electron usually gives a considerable degree of reactivity
to the free radical. ROS are well recognized for playing a dual
role as both deleterious and beneficial species (Valko et al., 2007).
The harmful effect of free radicals occurs in biological systems
when there is an overproduction of ROS on one side and a deficiency
of enzymatic and non-enzymatic antioxidants on the other.
Overproduction of ROS can be an important mediator of damage
to cell structures, including lipids and membranes, proteins,
and DNA (Blander, Oliveira, Conboy, Haigis, & Guarente, 2003;
Harman, 1993).
Vitamin E has a fundamental role in the normal metabolism of
all cells. It has biological antioxidant activity capable of terminating
chain reactions and it can chemically prevent lipid oxidation
* Corresponding author. Tel.: þ1 864 656 6246; fax: þ1 864 656 4395.
E-mail address: wwhtsd@clemson.edu (S. Whiteside).
0023-6438/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2010.06.032
LWT - Food Science and Technology 44 (2011) 24e28
Contents lists available at ScienceDirect
LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
a-tocopherol-loaded poly 3-caprolactone (PCL) nanoparticles were prepared by emulsion solvent evaporation
with ultrasonification technique. The influences of PCL concentration (3 and 5 g/100 mL), solvent
in the oil phase (methylene chloride (DCM) and methylene chloride: acetonitrile ¼ 50:50 (DCM:ACN)),
and ultrasonification time (1, 2, and 3 min) were investigated. Encapsulation efficiency (%) was calculated
by Duncan’s multiple rage tests and it decreased from 87.73 to 57.45 when ultrasonification time was
increased from 1 to 3 min. DCM as a solvent in the oil phase and 5 g/100 mL PCL showed better
encapsulation efficiency than DCM:ACN and 3 g/100 mL PCL. Particle mean size was decreased when
ultrasonification time was increased from 1 to 3 min. Nanoparticles with DCM as a solvent in the oil
phase had larger particle mean size than the particle with DCM:ACN. There were no significant differences
in particle mean size between two PCL concentrations. PCL with 3 g/100 mL concentration had
higher a-tocopherol loading (%) than 5 g/100 mL PCL. Overall, 5 g/100 mL PCL in DCM as solvent in the oil
phase with 3 min ultrasonification time showed the best encapsulation formulation.
Ó 2010 Elsevier Ltd. All rights reserved.
(Combs, 2008, pp. 181e212). a-tocopherol is the most abundant
lipid-soluble, chain-breaking antioxidant and it is the most biologically
active form of vitamin E compounds. The free radical
scavenging reactivity of the four tocopherols has been measured,
with the order of a-tocopherol > g-tocopherol > b-tocopherol > dtocopherol
(Lien, Ren, Bui, & Wang, 1999).
Many methods have been developed for preparing nanoparticles.
Commonly used methods of preparing nanoparticles
from biodegradable polymers include emulsion solvent evaporation
(Sahoo, Panyam, Prabha, & Labhasetwar, 2002; Scholes et al.,
1993), nanoprecipitation (Govender, Stolnik, Garnett, Illum, &
Davis, 1999; Leo, Brina, Forni, & Vandelli, 2004), salting out procedure
(Konan, Gurny, & Allemann, 2002), and a combined method
(Mccarron, Donnelly, & Marouf, 2006). The difference between
emulsion solvent evaporation and nanoprecipitation is that the
main phases in the emulsion solvent evaporation stay immiscible at
all times, only to be removed later by evaporation. Emulsion solvent
evaporation method involves two steps. The first step requires
emulsification of the polymer solution into an aqueous phase.
During the second step, the solvent used in the polymer solvent is
evaporated, inducing polymer precipitation as nanospheres (Reis,
Neufeld, Ribeiro, & Veiga, 2006).

In this research, poly 3-caprolactone (PCL) was used to encapsulate
a-tocopherol within nanospheres. PCL is semi-crystalline
biodegradable and biocompatible polyester with low glass transition
temperature and melting point (Pitt, 1990). It has been investigated
for drug delivery for several years and it is non-toxic and
non-mutagenic (Forrest, Zhao, Won, Malick, & Kwon, 2006; Mora-
Huertas, Fessi, & Elaissari, 2010). Moreover, it is considerably lower
cost than other biodegradable polyesters such as polyglycolide,
polylactide, and their copolymers.
The objective of this work was to entrap a-tocopherol within
PCL nanoparticles by O/W emulsion solvent evaporation with
ultrasonification method and to optimize the encapsulation
formulation. To achieve this goal, this study was designed to assess
the influence of formulation variables on the characteristics of
nanoparticles such as encapsulation efficiency, a-tocopherol
loading, particle size, zeta potential, and morphology. The formulation
variables were as follows: (1) solvent in the oil phase;
(2) concentration of PCL; (3) ultrasonification time.
2. Materials and methods
2.1. Materials
a-tocopherol (Mw 430.7 g/mol) was purchased from EMD
Bioscience (CA, USA). Poly 3-caprolactone (PCL, Mw 65,000 g/mol)
was purchased from SigmaeAldrich (MO, USA). Polyvinyl alcohol
(PVA, Mw 22,000 g/mol) was purchased from Acros organics (NJ,
USA). Methylene chloride (DCM), methanol, and acetonitrile (ACN),
all HPLC grade were purchased from J.T.Baker (USA). Phosphate
Buffered Saline (PBS, 10 liquid concentrate) was purchased from
EMD Bioscience (CA, USA).
2.2. Formulation of nanoparticles containing a-tocopherol
In this study, nanoparticles were prepared using an oil-in-water
emulsion solvent evaporation with ultrasonification technique
(Fig. 1) by modification of previous works (Kim, Hwang, Park, &
Park, 2005 and Konan et al., 2002). Three factors were statistically
examined, solvent in the oil phase, PCL concentration, and
ultrasonification time (Table 1).
In this procedure, specific amount of PCL (300 or 500 mg) was
dissolved in 10 mL of solvent (methylene chloride or methylene
chloride:acetonitrile ¼ 50:50) containing 10 mg of a-tocopherol.
A PVA (2g/100 mL) solution was prepared in PBS solution. The
PCL solution was added to 40 mL of the PVA solution. The total
mixture in 250 mL centrifuge bottle was then placed in an ice bath
and emulsified using a Branson Digital Sonifier (model 250, Connecticut,
USA) with 55 W of energy output for a specific time
(1, 2, and 3 min) to obtain an oil-in-water emulsion. Another 40 mL of
the PVA solution was then added to the emulsion. The final solution
was stirred for 12 h at 300 rpm on a magnetic stir plate to allow the
evaporation of methylene chloride and acetonitrile and to allow
the formation of the nanoparticles. The suspension was then
centrifuged at 4880 g for 20 min. The pellet was resuspended in
distilled water and centrifuged three more times at 1220 g for 20 min
each. These washing steps were performed to remove unencapsulated
PVA and a-tocopherol. The nanoparticles were collected and
frozen at 80 C for at least 2 h and subsequently freeze dried for 2
days. The freeze dried nanoparticles were stored at 4 C.
2.3. a-Tocopherol encapsulation efficiency (%) and loading (%)
Encapsulation efficiency was determined by an extraction
method. Dried nanoparticles (10 mg) were dissolved in 5 mL of
methylene chloride and 5 mL of distilled water. The mixture was
Y. Byun et al. / LWT - Food Science and Technology 44 (2011) 24e28 25
Fig. 1. Flow chart depicting the encapsulation process.
vigorously vortexed for 1 min and sonicated 5 min in order to
extract the a-tocopherol into the organic solution. Then, methylene
chloride was evaporated and replaced with methanol. The 2 mL of
organic solution was filtered and the a-tocopherol content of the
solution was analyzed by HPLC (Waters 1525 Binary HPLC pump,
USA).
The experiment was performed in triplicate and the encapsulation
efficiency was calculated using the ratio of the mass of
Table 1
Batch compositions used for constituting a-tocopheroleloaded nanoparticle.
Code PCL concentration
(g/100 mL)
3M1 3 DCM a
Solvent in the oil
phase
3M2 3 DCM 2
3M3 3 DCM 3
5M1 5 DCM 1
5M2 5 DCM 2
5M3 5 DCM 3
3MA1 3 DCM:ACN b
1
3MA2 3 DCM:ACN 2
3MA3 3 DCM:ACN 3
5MA1 5 DCM:ACN 1
5MA2 5 DCM:ACN 2
5MA3 5 DCM:ACN 3
a DCM: Methylene chloride, ACN: Acetonitrile.
b (DCM:ACN): DCM:ACN ¼ 50:50.
Ultrasonification
time (min)
1

26
Fig. 2. Effects of (a) solvent in the oil phase, (b) PCL concentration, and (c) ultrasonification
time on particle mean size (n ¼ 18 and LSD ¼ 110.15 for PCL concentration
and solvent in the oil phase, n ¼ 12 and LSD ¼ 134.91 for ultrasonification time,
p < 0.05).
a-tocopherol determined analytically to the mass of a-tocopherol
added during the formation process, as shown in Eq. (1). The atocopherol
loading (%) was calculated using the ratio of the mass of
a-tocopherol determined to the mass of total nanoparticle, as
shown in Eq. (2).
Encapsulation efficiency ð%Þ ¼
Mass of a tocopherol determined ðmgÞ
Mass of a tocopherol added ðmgÞ
a tocopherol loading ð%Þ ¼
Mass of a tocopherol determined ðmgÞ
Mass of total nanoparticle ðmgÞ
2.4. Particle mean size and zeta potential
100 ð1Þ
100 ð2Þ
A dilute suspension of nanoparticles was prepared in distilled
water. Particle mean size and size distribution were determined by
Zetasizer (Nano-ZS, Malvern Instrumet, UK). The surface charges on
nanoparticles were also examined by measuring their zeta potentials
using the Zetasizer.
2.5. Scanning electron microscopy
The morphology of the nanoparticles was examined by scanning
electron microscopy (S-4800 UHR FE-SEM, Hitachi high technologies
America, Inc.). Surfaces were prepared using platinum coating.
SEM images were taken at 3 kV with 5 k magnification and 10 mm
scale bar was used.
Y. Byun et al. / LWT - Food Science and Technology 44 (2011) 24e28
Fig. 3. Effecs of (a) solvent in the oil phase, (b) PCL concentration, and (c) ultrasonification
time on encapsulation efficiency (n ¼ 18 and LSD ¼ 7.61 for PCL concentration
and solvent in the oil phase, n ¼ 12 and LSD ¼ 9.32 for ultrasonification time,
p < 0.05).
Fig. 4. Effects of (a) solvent in the oil phase, (b) PCL concentration, and (c) ultrasonification
time on loading (n ¼ 18 and LSD ¼ 0.2106 for PCL concentration and
solvent in the oil phase, n ¼ 12 and LSD ¼ 0.2579 for ultrasonification time, p < 0.05).

2.6. Statistical analysis
Statistics were performed with the analysis of variance (ANOVA)
using SAS (version 9.1, SAS Institute Inc., Cary, NC, USA). Differences
among mean values were processed by Duncan’s multiple range
tests and the least significant difference (LSD). Significance was
defined at a level of p < 0.05.
3. Results and discussion
3.1. The effect of solvent on properties of nanoparticle
The solvent in the oil phase dissolves into the aqueous phase
and then, evaporates in the O/W emulsion solvent evaporation
method. The extent and speed of solvent transfer from the oil
phase into the aqueous phase depends on the solubility. The
solvent composition can be a key factor in controlling the solvent
removal rate and the final size of the microspheres (Maia &
Santana, 2004). In this study, the particle mean size of the
nanoparticles prepared by DCM:ACN as the solvent in the oil
phase was smaller than that prepared by DCM only (Fig. 2). The
water-miscible solvent quickly diffused out of the polymer solution.
Then, DCM was removed leading to hardening of the capsule
(Luong-Van, Grondahl, Nurcombe, & Cool, 2007). The water
solubility of ACN was greater than DCM and therefore the rate of
precipitation was higher than the rate with DCM. Due to the high
water solubility of ACN, it had higher diffusion rate before
hardening. This was the major reason for the smaller particle
mean size prepared by mixture of DCM and ACN (Kim et al.,
2005). In this study, encapsulation efficiency was increased
when DCM was used as the solvent in the oil phase (Fig. 3). By
increasing the particle mean size, the a-tocopherol diffusion into
the aqueous solution decreased because there was a longer
distance to a-tocopherol travel. Consequently, higher encapsulation
efficiency was associated with an increase in the particle
Y. Byun et al. / LWT - Food Science and Technology 44 (2011) 24e28 27
Fig. 5. SEM images of nanoparticles (a) 5M1 (b) 5M2 (c) 5M3.
mean size. There were no significant differences in a-tocopherol
loading (%) between the two solvents (Fig. 4).
3.2. The effect of polymer concentration on properties of
nanoparticle
Polymer concentration is also a key factor. In this study, two PCL
concentrations, 3 and 5 g/100 mL, were selected. Those concentrations
showed the best result in preliminary test. The encapsulation
efficiency was increased by increasing PCL concentration
from 3 to 5 g/100 mL (Fig. 3). By increasing the polymer concentration
in the organic phase, the viscosity of the solution was
increased (Ito, Fujimori, & Makino, 2007). Increasing viscosity can
decrease the a-tocopherol diffusion into the aqueous phase and
thus increase the a-tocopherol incorporation into the nanoparticles
(Song et al., 2008). Consequently, the encapsulation efficiency of
nanoparticles was increased by increasing the polymer concentration.
Conversely, a-tocopherol loading (%) was decreased by
increasing PCL concentration (Fig. 4). Increasing viscosity increased
the total mass of PCL in the nanoparticles. This decreased the ratio
of a-tocopherol to the total mass of nanoparticles, thus decreasing
a-tocopherol loading (%). It was observed that there were no
significant differences in particle mean size between two PCL
concentrations (Fig. 2).
3.3. The effect of ultrasonification time on properties of
nanoparticle
Ultrasonification time is another key factor. In this study, particle
mean size was decreased by increasing ultrasonification time from
1 to 3 min (Fig. 2). The increased time of ultrasonification led to the
formation of smaller nanoparticles. It was also observed that
encapsulation efficiency was decreased by increasing ultrasonification
time from 1 to 3 min (Fig. 3). Increasing the ultrasonification
time resulted in a reduction in the encapsulation
efficiency due to the decreasing particle mean size (Song et al., 2008).

28
Table 2
Properties of a-tocopheroleloaded nanoparticle.
Code Encapsulation
efficiency (%)
3M1 79.57 10.32 bc
3M2 65.22 6.78 cd
3M3 54.53 5.22 d
5M1 96.42 4.27 a
5M2 90.72 14.15 ab
5M3 90.95 6.15 ab
3MA1 79.65 7.89 bc
3MA2 50.39 4.06 d
3MA3 24.91 1.83 e
5MA1 95.29 12.52 a
5MA2 63.18 9.54 d
5MA3 59.41 9.08 d
Again, encapsulation efficiency is associated with the particle mean
size. There were no significant differences in a-tocopherol loading
(%) with respect to ultrasonification time (Fig. 4).
3.4. Overall properties of nanoparticles
The shape of nanoparticles was spherical and regular as visualized
in the SEM photographs (Fig. 5). There were agglomerates of
fragments when DCM:ACN was used as a solvent in the oil phase
(Figure was not shown).
Selecting the best formulation is a critical issue for further
research. In this research, the nanoparticle with higher encapsulation
efficiency (%) had higher total amount of particle than the
nanoparticle with higher loading (%). Therefore, higher encapsulation
efficiency (%) is more important factor than higher loading
(%) to select the best formulation (Table 2).
Encapsulation efficiency ranged from 25 to 96% depending on
the different factors (Table 2). Only four formulations showed over
90% of encapsulation efficiency; 5M1, 5M2, 5M3, and 5MA1
(Table 2). a-tocopherol loading (%) in the nanoparticles ranged from
2.12 to 3.51% (Table 2). It was observed that a-tocopherol loading
(%) of 5M3 was higher than that of 5M1, 5M2, and 5MA1. In this
research, less than 500 nm of particle mean size was considered
as an adequate particle mean size and less than 0.5 of polydispersity
was considered as a good size distribution. Particle mean
size ranged from 247 to 1070 nm (Table 2). 5M3 had 369 nm
particle mean size with 0.27 polydispersity and 5M2 had 448 nm
particle mean size with 0.40 polydispersity. On the other hand,
5M1 and 5MA1 had particle mean size above 700 nm with 0.60
polydispersity.
4. Conclusion
a-tocopherol
loading (%)
3.01 0.38 ab
3.21 0.22 a
3.06 0.25 a
2.26 0.14 c
2.12 0.42 c
2.50 0.28 bc
3.51 0.35 a
3.20 0.20 a
3.19 0.17 a
2.41 0.49 c
2.12 0.34 c
2.31 0.32 c
Results are expressed as the mean SD (n ¼ 3).
aei The different letters within same column differ significantly (p < 0.05).
Total amount of
particle (mg)
264.33 2.52 d
203.00 16.64 fg
178.33 2.89 gh
426.33 10.02 a
430.67 22.14 a
365.67 33.86 b
226.67 2.89 ef
158.00 20.22 h
78.33 7.64 i
400.33 35.70 a
298.67 13.58 c
257.33 5.03 de
a-tocopherol-loaded PCL nanoparticles as a free radical scavenger
were prepared by O/W emulsion solvent evaporation with
ultrasonification technique. It has been shown that PCL concentration,
solvent in the oil phase, and ultrasonification time all
significantly affected the encapsulation efficiency (%). In contrast,
solvent in the oil phase and ultrasonification time did not significantly
affect a-tocopherol loading (%) and PCL concentration did
not significantly affect particle mean size.
Overall, 5 g/100 mL PCL in DCM as the solvent in the oil phase
with 3 min ultrasonification time showed good encapsulation
efficiency (%), smaller particle mean size with good polydispersity,
well-shaped particle, and high a-tocopherol loading (%). Due to
these results, this formulation was selected for further research.
Y. Byun et al. / LWT - Food Science and Technology 44 (2011) 24e28
References
Particle mean
size (nm)
1070 55.75 a
908.23 4.71 b
370.23 14.35 e
767.83 46.30 c
448.87 0.85 d
368.03 3.47 e
399.50 11.07 e
316.20 3.92 f
247.70 1.65 g
733.17 27.80 c
392.77 12.42 e
376.10 7.08 e
Polydispersity
(PI)
0.70 0.04 a
0.70 0.01 a
0.28 0.04 ef
0.59 0.02 b
0.40 0.02 c
0.27 0.03 ef
0.37 0.01 cd
0.25 0.01 f
0.10 0.04 g
0.60 0.04 b
0.33 0.02 de
0.29 0.08 ef
Zeta potential
(mV)
14.73 0.61 g
12.83 0.29 f
10.57 0.21 bc
11.97 0.49 ef
11.77 0.81 de
12.13 0.23 ef
10.87 0.72 cd
9.83 0.12 b
7.70 0.16 a
16.23 0.74 h
15.23 1.10 gh
15.70 0.26 gh
Blander, G., Oliveira, R. M., Conboy, C. M., Haigis, M., & Guarente, L. (2003). Superoxide
dismutase 1 knock-down induces senescence in human fibroblasts.
The Journal of Biological Chemistry, 278, 38966e38969.
Combs, G. F. (2008). The vitamins: fundamental aspects in nutrition and health.
London: Elsevier Academic Press.
Forrest, M., Zhao, A., Won, C., Malick, A., & Kwon, G. (2006). Lipophilic prodrugs of
Hsp90 inhibitor geldanamycin for nanoencapsulation in poly(ethylene glycol)b-poly(3-caprolactone)
micelles. Journal of Controlled Release, 116, 139e149.
Govender, T., Stolnik, S., Garnett, M., Illum, L., & Davis, S. (1999). PLGA nanoparticles
prepared by nanoprecipitation: drug loading and release studies of a water
soluble drug. Journal of Controlled Release, 57, 171e185.
Harman, D. (1993). Free radical involvement in aging, pathophysiology and therapeutic
implications. Drugs and Aging, 3, 60e80.
Ito, F., Fujimori, H., & Makino, K. (2007). Incorporation of water-soluble drugs in
PLGA microspheres. Colloids and Surfaces B: Biointerfaces, 54, 173e178.
Kim, B. K., Hwang, S. J., Park, J. B., & Park, H. J. (2005). Characteristics of felodipinelocated
poly(3-caprolactone) microspheres. Journal of Microencapsulation, 22,
193e203.
Konan, Y., Gurny, R., & Allemann, E. (2002). Preparation and characterization of
sterile and freeze-dried sub-200 nm nanoparticles. International Journal of
Pharmaceutics, 233, 239e252.
Leo, E., Brina, B., Forni, F., & Vandelli, M. (2004). In vitro evaluation of PLA nanoparticles
containing a lipophilic drug in water-soluble or insoluble form.
International Journal of Pharmaceutics, 278, 133e141.
Lien, E., Ren, S., Bui, H., & Wang, R. (1999). Quantitative structureeactivity relationship
analysis of phenolic antioxidants. Free Radical Biology and Medicine, 26,
285e294.
Luong-Van, E., Grondahl, L., Nurcombe, V., & Cool, S. (2007). In vitro biocompatibility
and bioactivity of microencapsulated heparan sulfate. Biomaterials, 28,
2127e2136.
Maia, J., & Santana, M. (2004). The effect of some processing conditions on the
characteristics of biodegradable microspheres obtained by an emulsion solvent
evaporation process. Brazilian Journal of Chemical Engineering, 21, 1e12.
Mccarron, P., Donnelly, R., & Marouf, W. (2006). Celecoxib-loaded poly(D, L-lactideco-glycolide)
nanoparticles prepared using a novel and controllable combination
of diffusion and emulsification steps as part of the salting-out procedure.
Journal of Microencapsulation, 23, 480e498.
Mora-Huertas, C., Fessi, H., & Elaissari, A. (2010). Polymer-based nanocapsules for
drug delivery. International Journal of Pharmaceutics, 385, 113e142.
Pitt, C. G. (1990). Poly 3-caprolactone and its copolymer. In M. Chasin, & R. Lange
(Eds.), Biodegradable polymers as drug delivery systems (pp. 71e120). New York:
Marcel Dekker.
Reis, C., Neufeld, R., Ribeiro, A., & Veiga, F. (2006). Nanoencapsulation 1. Methods for
preparation of drug-loaded polymeric nanoparticles. Nanomedicine: Nanotechnology,
Biology and Medicine, 2, 8e21.
Sahoo, S., Panyam, J., Prabha, S., & Labhasetwar, V. (2002). Residual polyvinyl alcohol
associated with poly(D, L-lactide-co-glycolide) nanoparticles affects their physical
properties and cellular uptake. Journal of Controlled Release, 82,105e114.
Scholes, P., Coombes, A., Illum, L., Davis, S., Vert, M., & Davies, M. (1993). The
preparation of sub-200 nm poly(lactide-co-glycolide) microspheres for sitespecific
drug delivery. Journal of Controlled Release, 25, 145e153.
Song, X., Zhao, Y., Wu, W., Bi, Y., Cai, Z., Chen, Q., et al. (2008). PLGA nanoparticle
simultaneously loaded with vincristine sulfate and verapamil hydrochloride:
systematic study of particle size and drug entrapment efficiency. International
Journal of Pharmaceutics, 350, 320e329.
Valko, M., Leibfritz, D., Moncol, J., Cronin, M., Mazur, M., & Telser, J. (2007). Free
radicals and antioxidants in normal physiological functions and human disease.
The International Journal of Biochemistry and Cell Biology, 39, 44e84.