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DOI 10.1007/s00217-010-1341-4
ORIGINAL PAPER
Received: 12 March 2010 / Revised: 9 July 2010 / Accepted: 27 July 2010 / Published online: 10 August 2010
Springer-Verlag 2010
Introduction
Tea (Camellia sinensis, Theaceace) is a popular consumed
beverage in the world. Tea polyphenols are one of the main
bioactive components of tea leaves, which include catechines, flavanols, flavanones, phenolic acids, glycosides
and the aglycons of plant pigments [1]. Tea polyphenols,
used in food industry and medicine for a long time, have
attracted much attention in recent years, due to their biological activities, such as antioxidant and antitumor activities [2]. Consumption of tea polyphenols has been linked
to the prevention of a variety of cancers using animal
models, including prevention of cancers of the skin,
digestive tract, liver, bladder and prostate [3]. On the other
hand, tea polyphenols are not stable, and their bioavailability limits the biological activity in vivo. In humans,
modest transient increases in plasma antioxidant capacity
have been demonstrated following the consumption of tea
and green tea catechins [4]. The antioxidant activity of tea
polyphenols decreased dramatically when it was exposed
to alkaline pH as in the human intestine. The phenolic
hydroxyl of tea polyphenols could be easily oxidized and
lost biological activity, thus their oral bioavailability was
known to be low [5]. Nakagawa et al. [6] had also shown
that the concentration of tea polyphenols in plasma
decreased rapidly.
It had been reported that the biological activity of
tea polyphenols might depend on the form of their
administration [7]. Therefore, it was essential to design an
effective delivery system for improving tea polyphenols
bioavailability.
Nanoparticles were under consideration for oral drug
delivery by stabilizing and ensuring biological activity
during transit through the gastrointestinal tract and by
facilitating absorption and delivery to the target site [8].
123
918
123
919
EE%
2.00
3.00
4.00
0.80
1.20
1.60
5.00
10.00
15.00
Constraints
Dependent variables
Y1 = particle size (nm)
Minimize
Maximize
Experimental design
BoxBehnken design was generally selected to optimize
the formulation parameters and evaluate the main effects
for nanoclusters formulation ingredients since it required
few runs in case of three or four variables [8]. In this
study, a 3-factor, 3-level BoxBehnken design was used
to optimize nanoclusters formulation with carboxymethyl
chitosan concentration (X1), chitosan hydrochloride concentration (X2) and amount of tea polyphenols (X3) as the
independent variables with low, medium and high concentration values described in Table 1. The mathematical
relationship of two responses particle size (Y1) and
entrapment efficacy (Y2) and three independent variables
(Xi) were modeled by a second-order polynomial function
as follows:
Y b0 b1 X1 b2 X2 b3 X3 b12 X1 X2 b13 X1 X3
b23 X2 X3 b11 X12 b22 X22 b33 X32
123
920
Independent variables
Responses
Ya2
X2
3.00
0.80
5.00
358
44.4
2.00
1.20
5.00
746
64.0
2.00
1.60
10.00
1079
72.7
4.00
0.80
10.00
403
61.7
4.00
1.20
15.00
349
80.3
3.00
1.60
5.00
557
69.2
2.00
1.20
15.00
1019
57.6
4.00
1.60
10.00
472
69.4
3.00
0.80
15.00
307
64.4
10
2.00
0.80
10.00
884
45.5
11
3.00
1.60
15.00
867
62.2
12
4.00
1.20
5.00
472
70.2
13
3.00
1.20
10.00
347
80.9
14
15
3.00
3.00
1.20
1.20
10.00
10.00
331
407
80.6
83.0
X3
Ya1
X1
123
Source
Sum of squares
Model
984000
A-Carboxymethyl chitosan
B-Chitosan hydrochloride
df
Mean square
F value
P value
109300
14.42
0.0045a
516100
516100
68.06
0.0004a
130800
130800
17.25
0.0089a
C-Tea polyphenols
20910.13
20910.13
2.76
0.1577
AB
3969.00
3969.00
0.52
0.5018
AC
39204.00
39204.00
5.17
0.0721
BC
32580.25
32580.25
4.30
0.0929
A2
205700
205700
27.13
0.0034a
B2
46144.16
46144.16
6.09
0.0568
8790.01
8790.01
1.16
0.3308
Residual
37915.92
7583.18
Lack of fit
34705.25
11568.42
7.21
0.1243
Pure error
3210.67
1605.33
Cor. total
1022000
14
921
123
922
Table 4 The analysis of
variance table for encapsulation
efficiency as the response (Y2)
Source
Sum of squares
Model
1927.43
A-Carboxymethyl chitosan
B-Chitosan hydrochloride
214.16
16.44
0.0033a
218.40
218.40
16.76
0.0094a
413.28
413.28
31.72
0.0024a
34.86
34.86
2.68
0.1628
95.06
7.30
0.0427a
AC
68.06
68.06
5.22
0.0710
BC
182.25
182.25
13.99
0.0134a
115.79
115.79
8.89
0.0308a
680.42
680.42
52.23
0.0008a
228.98
228.98
17.58
0.0086a
Residual
65.14
13.03
Lack of fit
61.72
20.57
12.03
0.0777
Pure error
3.42
1.71
Cor. total
1992.57
14
123
P value
95.06
5% significance level
F value
AB
Mean square
C-Tea polyphenols
df
923
123
924
also interfere with the chitosan cross-linking of tea polyphenols molecules so that entrapped tea polyphenols was
leaked more easily during the nanoclusters preparation.
Optimization and model validation
The optimum nanoclusters for encapsulating tea polyphenols were selected based on the formulation parameters. By
analysis of response surface plots obtaining by Design
Expert software and numerical solution for Eqs. 3, 4 and
constraints, the optimum values for independent variables
in uncoded (actual) units to minimize the particle size and
maximize the encapsulation responses were that the concentration of carboxymethyl chitosan and chitosan hydrochloride was 3.63, 1.19 mg/mL and amount of tea
polyphenols was 10.94 mg. According to the condition, the
particle size and encapsulation efficacy predicted by the
model calculated as 292 nm and 83.1%, respectively. For
validation of the model, five experiments were performed
by using the optimum condition as mentioned previously.
The optimized nanoclusters formulation had a mean particle size of 301 nm, and tea polyphenols entrapment
efficiency was added up to 83.7%. The formulation of
nanoclusters with solution described earlier showed the
validity of the BoxBehnken design for the optimization of
nanoparticle formulation demonstrated by experimental
values similar to predicted values within 5% of predicted
error. The perfect agreement between the observed values
and the values predicted by the equation confirmed the
statistically significance of two models as well as their
adequate precision for the prediction of optimum conditions in the domain of levels chosen for the independent
variables.
Conclusion
It was confirmed that BoxBehnken design is a very useful
tool in preparation and optimization of tea polyphenolsloaded chitosan nanoclusters studies. The optimal parameters like carboxymethyl chitosan and chitosan hydrochloride
concentration and amount of tea polyphenols that could
affect the particle size and encapsulation efficacy of
nanoclusters were identified. The nanoclusters polyionic
complexation prepared using two biomaterials carboxymethyl chitosan and chitosan hydrochloride could successfully be used to encapsulate tea polyphenols. However, the
absorption and biological activity of tea polyphenols-loaded
chitosan nanoclusters are still unknown. Therefore, our
future study will focus on the application of chitosan
123
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