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Eur Food Res Technol (2010) 231:917924

DOI 10.1007/s00217-010-1341-4

ORIGINAL PAPER

Response surface methodology in the optimization of tea


polyphenols-loaded chitosan nanoclusters formulations
Jin Liang Feng Li Yong Fang Wenjian Yang
Xinxin An Liyan Zhao Zhihong Xin
Qiuhui Hu

Received: 12 March 2010 / Revised: 9 July 2010 / Accepted: 27 July 2010 / Published online: 10 August 2010
Springer-Verlag 2010

Abstract Response surface methodology was used to


optimize tea polyphenols-loaded chitosan nanoclusters
preparation conditions, including carboxymethyl chitosan
concentration, chitosan hydrochloride concentration and
amount of tea polyphenols. The responses particle size and
entrapment efficiency of nanoclusters were studied. The
optimum conditions of carboxymethyl chitosan concentration, chitosan hydrochloride concentration and amount
of tea polyphenols were found to be 3.63, 1.19 and
10.94 mg/mL, respectively. The optimized particle size
was 301 nm, and entrapment efficacy of nanoclusters was
added up to 83.7%. The results demonstrated that Box
Behnken design methodology was an effective way to
obtain the optimal formulation of tea polyphenols-loaded
chitosan nanoclusters, and the nanoclusters complexation
synthesizing through ionic gelation between carboxymethyl chitosan and chitosan hydrochloride was good
biomaterials, which could be successfully used to encapsulate tea polyphenols.
Keywords Tea polyphenols  Carboxymethyl chitosan 
Chitosan hydrochloride  Nanoclusters  BoxBehnken
design

J. Liang  F. Li  Y. Fang  W. Yang  X. An  L. Zhao 


Z. Xin  Q. Hu (&)
College of Food Science and Technology,
Nanjing Agricultural University,
Nanjing 210095, Peoples Republic of China
e-mail: qiuhuihu@njau.edu.cn

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].

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Chitosan, the deacetylated derivative of chitin, as a natural


biopolymer specifically in drug delivery had attracted
particular interest due to their desirable biocompatible,
biodegradable, hydrophilic and protective properties [9]. It
had been applied broadly as a functional biopolymer in
food and pharmaceutics. Especially, due to their subcellular and submicrometer size, chitosan nanoparticles could
penetrate deep into tissues through fine capillaries and
cross the fenestration present in the epithelial lining. This
allowed efficient delivery of therapeutic agents to target
sites in the body [10]. Therefore, there had been considerable interest in developing chitosan nanoparticles as
effective drug delivery devices. Generally, chitosan nanoparticles as drug carriers were synthesized using ionic
gelation method [11, 12]. The interaction between biodegradable cationic and anionic biopolymers leaded to the
formation of polyionic hydrogels, which had demonstrated
favorable characteristics for drug entrapment and delivery
[13].
Nanoclusters are tiny nanocomposites. These clusters act
as microscopic transport vehicles can be formed through
self-assembly of nanoparticles in solution [14]. Carboxymethyl chitosan and chitosan hydrochloride are two different water-soluble chitosan, and they have received much
attention with anionic and cationic, respectively, and been
studied for drug entrapment and delivery [15, 16]. Carboxymethyl chitosan and chitosan hydrochloride can form
nanoclusters complexation through ionic gelation via
interactions between the carboxyl groups of carboxymethyl
chitosan and the amine groups of chitosan hydrochloride in
aqueous solution. The polyionic complexation can protect
the encapsulant, has biocompatible and biodegradable
characteristics and limits the release of encapsulated
materials more effectively. It is also thought to contribute to
longer in vivo circulation times and allow encapsulation of
water-soluble biomolecules [10, 17].
The physicochemical properties of nanoparticles are
important in determining the physiological functions and
stability of chitosan nanoparticles loaded with drugs. The
particle size of nanoparticles is one of the most significant
determinants in mucosal and epithelial tissue uptake and
intracellular trafficking. The unique character with positive
charge and small particle size of chitosan nanoparticles are
responsible for their in vivo efficacy [18]. Some reports
were found in the literature on controlling the fabrication
parameters to modulate the physicochemical aspects of
chitosan nanoparticles for the delivery of macromolecules
such as genes and proteins in therapeutics [19, 20]. However, little information on chitosan nanoparticles loaded
with bioactive polyphenolic antioxidant was available [21].
Moreover, considering the function in metabolic processes
as well as properties of tea polyphenols manifested in vitro,

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Eur Food Res Technol (2010) 231:917924

entrapment using chitosan nanoparticles was expected to


protect tea polyphenols from interactions with the food
matrix and also to allow controlled release in the gastrointestinal tract. Hence, in this study, chitosan nanoclusters
complexation prepared by ionic gelation between carboxymethyl chitosan and chitosan hydrochloride was used
as encapsulant materials for entrapment of tea polyphenols.
Response surface methodology was used to determine the
optimum level of each factor by building a mathematical
BoxBehnken experiment design model. The responses
particle size and entrapment efficiency of nanoclusters
were studied, and the zeta potential and morphology of
nanoclusters also were characterized. The most significant
factor was then selected, and the optimization of particle
size and entrapment efficacy could modulate by adjusting
the formation parameters.

Materials and methods


Materials
Green tea was purchased from Fukunfuxi Tea Company
(Yixing, Wuxi, Jiangsu, China), and tea polyphenols with
93% purity were obtained from the green tea by extraction
with 50% ethanol [22] and then purification with H1020
resins (Nankai University chemical plant, Tianjin, China)
[23]. Carboxymethyl chitosan (degree of deacetylation
83%) and chitosan hydrochloride (degree of deacetylation
85%) were purchased from Haidebei Marine Bioengineering Company (Jinan, Shandong, China). Other reagents
were of analytical grade.
Preparation of nanoclusters
Tea polyphenols-loaded chitosan nanoclusters were prepared by an ionic interaction method, performed according
to the following procedure [10]: Carboxymethyl chitosan
and chitosan hydrochloride were dissolved in distilled
water and prepared into different concentrations (as
Table 1 showed) aqueous solution by adding a certain
amount of tea polyphenols into carboxymethyl chitosan
solution. As a consequence, the addition of chitosan
hydrochloride solution (30 mL) was dropped slowly into
tea polyphenols and carboxymethyl chitosan mixture
solution (12 mL) with stirring at room temperature and
continuous stirring for 30 min. The formation of nanoclusters started spontaneously via the ionic gelation mechanism. The nanoclusters suspensions were immediately
subjected to further analysis and applications. The nonloaded nanoclusters without tea polyphenols were also
prepared as control.

Eur Food Res Technol (2010) 231:917924

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polyphenols-loaded chitosan nanoclusters was calculated


using Eq. 1:

Table 1 Variables used in BoxBehnken experimental design


Coded levels
-1

EE%

Independent variables symbol


Carboxymethyl chitosan (mg/mL) X1

2.00

3.00

4.00

Chitosan hydrochloride (mg/mL) X2

0.80

1.20

1.60

Tea polyphenols (mg) X3

5.00

10.00

15.00

Constraints
Dependent variables
Y1 = particle size (nm)

Minimize

Y2 = encapsulation efficiency (%)

Maximize

Particle size, zeta potential and morphology


Dynamic light scattering (DLS) (Brookhaven Instruments
Corporation, Holtsville, NY) was used to measure the
average particle size. Zeta potential was performed on a
Zeta sizer Nano-ZS (Malvern Instruments, England, UK)
on the basis of DLS techniques. All measurements were
taken in triplicate. Morphological examination of the
nanoclusters was performed by transmission electron
microscopy (TEM) (JEOL H-7650, Hitachi High-Technologies Corporation, Tokyo, Japan).
Determination of entrapment efficiency
Tea polyphenols-loaded chitosan nanoclusters suspensions
were centrifuged at 15,000 rpm at 4 C for 30 min. The
free tea polyphenols in the clear supernatant were determined in triplicate by the tea polyphenols colorimetric
assay method [24] described below.
The clear supernatant was transferred into 50-mL volumetric flask and achieved to the fixed scale with distilled
water. To determine tea polyphenols concentration of the
sample, 5 mL aqueous solution of tea polyphenols was
taken from the sample and added into a 25-mL volumetric
flask, then 5 mL ferrous tartrate tetrahydrate solution
(containing 1 g ferrous sulfate and 5 g potassium sodium
tartrate tetrahydrate dissolved in 1,000 mL water) was
also added into the volumetric flask and made up to
25 mL with potassium phosphate buffer solution (pH 7.5).
Several minutes were required for the color to develop.
With a blank solution (without tea polyphenols) as reference solution, the absorbance at 540 nm in a 10-mm
quartz cell was used to calculate the amount of free tea
polyphenols according to the prepared standard equation
of tea polyphenols solution (y = 0.0322x - 0.0156,
R2 = 0.9993). The entrapment efficiency (EE) of tea

Total tea polyphenols  Free tea polyphenols


Total tea polyphenols
 100%
1

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

where Y was the predicted response, b0, intercept, b1, b2


and b3, linear coefficients, b11, b22 and b33, squared coefficients and b12, b13 and b23, the interaction coefficients of
the equation and X1, X2 and X3 were the independent
variables. Using this equation, it was possible to evaluate
the linear, quadratic and interactive effects of the independent variables on the responses appropriately.
Optimization data analysis and model validation
The software Design Expert (Version 7.1.6, Stat-Ease Inc.,
Minneapolis, MN, USA) was employed for generation and
evaluation of the statistical experimental design. The
analysis of variance (ANOVA) provision available in the
software was used to establish the statistical validation of
the polynomial equations generated. A total of 15 runs with
triplicate center points were generated by BoxBehnken
design. All the responses observed were simultaneously
fitted to first-order-, second-order- and quadratic-models
and were evaluated in terms of statistically significant
coefficients and R2 values. Various feasibility and grid
searches were conducted over the experimental domain to
find the compositions of the optimized nanoclusters formulations. Three-dimensional response surface plots were
provided by the Design Expert software.

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Eur Food Res Technol (2010) 231:917924

Results and discussion


Preparation and characterization of nanoclusters
Tea polyphenols-loaded chitosan nanoclusters were prepared by ionic gelation process using BoxBehnken
experiment design and performed to determine the relationship between carboxymethyl chitosan, chitosan
hydrochloride and tea polyphenols on response variables
for the optimization of nanoclusters formulation.
The observed responses for particle size and entrapment
efficiency were presented in Table 2. It could be observed
that the particle size ranging from 307 to 1079 nm was
obtained through different 15 designed experiments in this
study. The mean diameter of nanoclusters was mainly
dependent upon concentration of carboxymethyl chitosan
and chitosan hydrochloride. Comparing batches 3 and 9,
the particle size increased with increasing chitosan hydrochloride concentration and decreased with increasing carboxymethyl chitosan concentration. It was likely that
intramolecular cross-linking of individual carboxymethyl
chitosan chains led to a pregel state and compacted coiled
structure at a lower chitosan hydrochloride concentration.
However, in situations of chitosan hydrochloride saturation
at higher concentration, lateral association of a number of
carboxymethyl chitosan chains led to larger nanoparticles
complexation structures. Regulating formulation conditions of nanoclusters, especially concentration of carboxymethyl chitosan and chitosan hydrochloride, the range
of particle size had a corresponding change. Similar
research had been also reported that chitosan complexation
Table 2 Composition and responses in BoxBehnken design
Batches

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

Values are means of three determinations

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contributed to the size of the alginate nucleus in proportion


to its initial diameter, since larger nuclei had more carboxylic groups to interact with amino groups of chitosan
[25].
Zeta potential was an important parameter to reflect
the physicochemical and biological stability of nanoclusters in suspension. In general, if the value of the particle zeta potential was large, the colloidal system was
stable. Conversely, the particle zeta potential was relatively small, which showed the colloidal system was in
agglomerate condition [26]. In this study, zeta potential of
all the nanoclusters prepared was measured ranging from
-40 to ?65 mV (data not shown). The zeta potential of
tea polyphenols-loaded chitosan nanoclusters prepared
at triplicate center points was about ?30 mV; this relatively high positive value showed higher electrostatic
stabilization of nanoclusters in suspension, demonstrating
the colloidal system has lower aggregation tendency and
relatively stable condition.
Entrapment efficiency of tea polyphenols was influenced
by the three preparation parameters. High tea polyphenols
entrapment with value above 83% was obtained with
higher concentration of carboxymethyl chitosan and
chitosan hydrochloride, while maintaining the other factors
at constant levels. Hu et al. [10] reported that the encapsulation efficiency of tea catechins nanoclusters produced
by chitosan and tripolyphosphate increased linearly with
the increase in chitosan concentration in a certain range.
Moreover, tea polyphenols entrapment was affected by the
addition amount of tea polyphenols as shown in Table 2,
where the entrapment efficiency was increased with
increasing amount of tea polyphenols, such as batches 1
and 9, batches 5 and 12. Some exceptions were seen when
comparing batches 2 and 7 or batches 6 and 11; the values
of entrapment efficiency were decreased with increasing
amount of tea polyphenols. It was possible that the addition
of chitosan solution at the nanoclusters suspension affected
the strength of electrostatic interaction between tea polyphenols and chitosan nuclei, and consequently, enhanced
or reduced the tea polyphenols entrapment efficiency.
Morphology of nanoclusters
With TEM studied, the structure of tea polyphenols-loaded
chitosan nanoclusters was illustrated in Fig. 1. The report
about chitosan nanoparticles prepared in different conditions presented a very homogeneous morphology and
showed a quite uniform particle size distribution and a
rather spherical shape [27]. However, Fig. 1 showed the
morphology of tea polyphenols-loaded chitosan nanoclusters was completely not spherical in shape and had rough
surface. In addition, the diameter of the combined tea
polyphenols-loaded chitosan nanoclusters was relatively

Eur Food Res Technol (2010) 231:917924

Fig. 1 TEM image of tea polyphenols-loaded chitosan nanoclusters

larger than non-loaded nanoclusters with the same prepared


parameters, which showed that the cross-linked chitosan
nanoclusters after loading with drugs resulted in a broader
size distribution [28, 29]. It was possible that the addition
of tea polyphenols changed particles cross-link between
chitosan chains and tea polyphenols and increased in particle size after forming nanocluster in aqueous solution,
which showed tea polyphenols-loaded chitosan nanoclusters with irregular shape.
BoxBehnken design and response surface
methodology
The effects of three nanoclusters preparation conditions,
including concentration of carboxymethyl chitosan, chitosan
Table 3 The analysis of
variance table for particle size
as the response (Y1)

R2 = 0.96, R2(adjust) = 0.90


5% significance level

hydrochloride and tea polyphenols in gelling medium,


on the responses particle size and entrapment efficiency, were examined using BoxBehnken experimental
design methodology (Table 2). Statistical analysis of the
experimental data was used to establish the best-fitted
models for the independent variables. To choose the best
model matched with the data, the analysis of variance by
calculating F-value was employed (Tables 3, 4). As a
result, for both responses, a full quadratic second-order
polynomial equation was fitted to the data appropriately.
The lack of fit F value of 0.1243 and 0.0777 implied
that the lack of fit was not significant in both responses,
due to the pure error and showed adequacy of the
models.
As Table 3 showed, the fitness of the model was
examined by determination coefficient (R2 = 0.96), which
indicated that the sample variation of more than 96% was
attributed to the variables. In addition, value of lack of fit
F value and lack of fit P value was found to be 7.21 and
0.1243, respectively, which implied that the lack of fit
was insignificant relative to the pure error. Insignificant
lack of fit made the model fit. Therefore, the Eq. 3 fitted
to the particle size (Y1) was presented, respectively, as
follows:
Y1 3627:45  1377:75 X1  1572:18 X2  23:56 X3
 78:75 X1 X2  19:80X1 X3 45:12 X2 X3
236:04 X12 698:69X22 1:95X32

When entrapment efficacy was considered as the


response, the results were much better. R2 and R2 (adjust)
of this model were 0.97 and 0.91, respectively (Table 4).
The mathematical relationship between entrapment
efficacy (Y2) and the nanoclusters preparation conditions
(Xi) obtained from the regression analysis using Eq. 4 was
given by:

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)

Eur Food Res Technol (2010) 231:917924

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

Y2  223:61 45:20X1 291:91X2 8:299X3


 12:19X1 X2 0:83X1 X3  3:38X2 X3  5:60X12
 84:84X22  0:32X32

The ANOVA of the regression model demonstrated that


the model was highly significant and adequate (R2 = 0.97).
The linear terms of carboxymethyl chitosan and chitosan
hydrochloride most significantly influenced entrapment
efficacy, and the effects were both negative.
The response surface plots were used for interpretation
of the interaction effects of two independent variables on
the responses or dependent variables when a third factor
was kept at constant level. The three-dimensional response
surfaces plotted by Design Expert software of the independent variables on both responses (Y1 and Y2) were
shown in Figs. 2 and 3.
Response surface plots showing the effect of carboxymethyl chitosan, chitosan hydrochloride and tea polyphenols on particle size could be seen in Fig. 2. In each plot,
the interaction of two variables was investigated simultaneously, while the third one was in its middle level value.
As a result, Fig. 2a showed the non-linear synergistic
relationship between the two independent variables (X1
and X2) on particle size. The particle size decreased with
increasing carboxymethyl chitosan concentration and
decreasing chitosan hydrochloride concentration within a
certain range. Moreover, the concentration of carboxymethyl chitosan seems to have more pronounced effect on
the particle size than chitosan hydrochloride. Woitiski et al.
[8] had also obtained nanoparticles by ionotropic pregelation and reported that the particle size decreased with
decreasing concentration of both calcium chloride and
chitosan and increasing drug concentration. As shown in
Fig. 2b, the minimum levels of particle size were measured

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P value

95.06

5% significance level

F value

AB

Mean square

C-Tea polyphenols

R2 = 0.97, R2(adjust) = 0.91

df

near higher levels of carboxymethyl chitosan. It also could


be inferred the carboxymethyl chitosan concentration
showed a greater influence on particle size than the tea
polyphenols. Figure 2c depicted the combined effect of
chitosan hydrochloride concentration and amount of tea
polyphenols on the particle size. The curvature in both
variables (X2 and X3) was quite obvious different. The
particle size was influenced mainly by chitosan hydrochloride concentration, which indicated the statistical
significance of quadratic coefficients of chitosan hydrochloride in the model, while tea polyphenols seemed to have
no significant effect on the particle size.
The convex shape of the plot shown in Fig. 3a indicated
that the optimum value (maximum value) for the response
in the range of variables was studied. The entrapment
efficacy increased when both carboxymethyl chitosan and
chitosan hydrochloride increased to reach a maximum and
then decreased at the combination of the high level of
variables. This observation may be caused by decreasing
capacity of nanoclusters to encapsulate tea polyphenols.
Reducing the carboxymethyl chitosan could cause
decreasing electrostatic attraction, and increasing chitosan
hydrochloride could lead to aggregation, thus the optimum
condition needed to be found to obtain the highest
entrapment efficacy. Figure 3b clearly showed the nonlinear and negative effects of carboxymethyl chitosan and
tea polyphenols on entrapment efficacy. This was quite
expectable because the increase in the two factors could
make the chitosan-cross-linked network structure of the
nanoclusters denser with less void volume available for the
entrapment of tea polyphenols-loaded chitosan nanoclusters. Figure 3c showed the quadratic and negative effect of
chitosan hydrochloride and tea polyphenols on entrapment
efficacy. A small addition of chitosan hydrochloride to the

Eur Food Res Technol (2010) 231:917924

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Fig. 2 Response surface plots showing effect of a carboxymethyl


chitosan and chitosan hydrochloride, b carboxymethyl chitosan and
tea polyphenols and c chitosan hydrochloride and tea polyphenols on
particle size

Fig. 3 Response surface plots showing effect of a carboxymethyl


chitosan and chitosan hydrochloride, b carboxymethyl chitosan and
tea polyphenols and c chitosan hydrochloride and tea polyphenols on
encapsulation efficiency

gelling medium leaded to a little increase in the level of


entrapment efficacy, probably because of its shielding
effect on the nanoclusters. When more than a critical

amount of chitosan hydrochloride was added, however, the


level of entrapment efficacy decreased again. This was
because an excess of chitosan hydrochloride could not only
reduce the void volume of tea polyphenols gel network but

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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

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Eur Food Res Technol (2010) 231:917924

nanoclusters for oral tea polyphenols delivery, using an


animal test.
Acknowledgments This work was supported by the National
Natural Science Foundation of China (30871743), the 111 Project
of Education Ministry of China (B07030) and the National High
Technology Research and Development Program of China (2007
AA100403).

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