C. R. F. SOUZA and W. P.

OLIVEIRA
Department of Pharmaceutical Sciences, Laboratory of Pharmaceutical Processes FCFRP/USP, Av. do Caf, s/n.,
CEP 14040-930, Ribeiro Preto (SP), Brazil e-mail: wpoliv@fcfrp.usp.br
ABSTRACT
INTRODUCTION
RESULTS AND DISCUSSION
MATERIALS AND METHODS
CONCLUSIONS
REFERENCES
Experimental design and multi-response analysis are powerful tools in the
characterization and optimization of pharmaceutical processes. In this work, this kind of
analysis were used in the optimization studies of the extraction of active compounds from
Bauhinia forficata Link with hydroalcoholic solutions. Preliminary tests were performed in
order to select the extraction method to be used in the optimization studies. Three distinct
methods were analyzed: sonification, maceration and a heated stirred reactor, indicating the
later as the most efficient. Next, a three factors and three levels Box-Behnken design and multi-
response analysis were used for the extraction process optimization. The studied factors
includes the extraction temperature (30 to 70
o
C), the composition of the extracting solvent
|m
w
/m
et
| (0 to 1) and the ratio between plant to solvent mass (0.1 to 0.2). Polynomials models
relating the solids and the flavonoids content in the extract with the studied variables were
adjusted by non-linear regression. The optimization of the models allowed the determination of
the best extraction conditions: Temperature of 50
o
C, Plant to solvent mass of 0.2, the relation
between the water to ethanol mass of 0.039.
BOX, G.E.P. and BEHNKEN, D.W., Some a New Three Level Designs for the Study of Quantitative Variables. Techno metrics, v. 2,
p. 455-475, 1960.
BOX, G.E.P., HUNTER, W.G. and HUNTER, J.S., Statistics for Experimenters: an Introduction to Design, Data Analysis, and
Model Building, New York, John Wiley e Sons, 1978.
DERRINGER, G. and SUICH, R. Simultaneous Optimization of Several Response Variables. Journal of Quality Technology, v.12, n.
4, p. 214-219, 1980.
ACKNOWLEDGEMENTS
The authors acknowledge FAPESP for financial support.
A common problem in product development involves the selection of a set of conditions,
which will result in a product with a desirable combination of properties. This essentially is a
problem involving the simultaneous optimization of several response variables ( the desirable
combination of properties) which depend upon a number of independent variables or sets of
conditions. Experimental design techniques such as factorial design and multi-response
analysis are useful tools in the characterization of pharmaceutical processes by studying the
effects of variables affecting them and their possible interactions. The objective of this study
was select the extraction method and determine the effect of the rate mpl/msol, relation |mw/met|
and temperature of extraction, for optimization of the extraction of active compounds from
Brazilian medicinal plants. The Bauhinia forficata Link was used as model plant due to your
use in the traditional folk medicine in the treatment of diabetes.

Three distinct extraction methods were analyzed: sonification, maceration and a heated
stirred reactor, indicating the later as the most efficient. Official method for spectrometric
determination of flavonoids was used. The procedure includes hydrolysis of glycosides,
extraction of the total flavonoids aglycones contents with ethyl acetate and developing color
with AlCl3. The absorbance is measured at 425 nm.
A three-factor, three-level Box-Behnken (1960) design was used. This design permits the
construction of a second-order polynomial model to characterize or optimize a process with
small number of experiments. The model has the following form:

Y
i
= a
0
+a
1
X
1
+ a
2
X
2
+ a
3
X
3
+ a
4
X
1
X
2
+ a
5
X
2
X
3
+ a
6
X
1
X
3
+ a
7
X
2
1
+ a
8
X
2
2
+ a
9
X
2
3
+E
The factor levels were evenly spaced and coded for low, medium, and high settings as - 1,
0, + 1 respectively. Table 1 summarizes the factors and their levels.
Table 1 - Factors and their levels for Box-Behnken design.
Bauhinia forficataLink (Pata de vaca)
Table 2 show the experimental
design in a randomized form. The
statistical evaluation of the results
was carried out by analysis of
variance (ANOVA) using a
sotfware package Statistica

.
Finally the desirability function
was used for the optimization of the process. During the optimization of a multivariable process,
such as extraction, the responses have to be combined in order to produce a product of desired
characteristics (desirability function combines all the responses in one measurement).
Table 2 Box-Behnken design (randomized).
- The heated stirred reactor showed the most efficient extraction method;
- The factorial design Box-Behnken and multi-response analysis are powerful tools in the
characterization and optimization of extractive processes and other pharmaceutical process;
- The optimization of the models allowed the determination of the best extraction conditions:
temperature of 50
o
C, plant to solvent mass of 0.2, the relation between the water to ethanol
mass of 0.039 (alcohol 70%).
The ANOVA results showed that the factors m
pl
/m
sol
(o=0.05) and |m
w
/m
et
| (o=0.01) had
significant influence on the flavonoids content, and with less significance (o=0.10) the
interaction between the variables m
pl
/m
sol
and |m
w
/m
et
|. For the solids content, the significant
factors were m
pl
/m
sol
and |m
w
/m
et
| (o=0.01). The experimental results were fitted by the
following polynomial models:
Table 3 Experimental design and experimental results of
TF and Cs.

Factors Levels
Temperature (C) 30 50 70
|mw/met| 0 0.5 1
mpl/msol (g/g)
0.1 0.15 0.20
Coded Variables
Experiment
Temperature |mw/met| mpl/msol
1 -1 -1 0
2 1 -1 0
3 -1 1 0
4 1 1 0
5 -1 0 1
6 1 0 1
7 -1 0 -1
8 1 0 -1
9 0 -1 1
10 0 1 1
11 0 -1 -1
12 0 1 -1
13 0 0 0
14 0 0 0
15 0 0 0
EXP VARIABLES RESULTS
T
( C )
|mw/met|
( g/g )
mpl/msol
( g/g )

( g/cm
3
)
TF
( %gflav/g.sol )
Cs
( g/gext )
1 -1 -1 0 1.00 1.33 0.07 0.017 0.001
2 1 -1 0 1.00 2.55 0.15 0.021 0.005
3 -1 1 0 0.80 7.71 0.17 0.012 0.004
4 1 1 0 0.81 6.92 0.88 0.018 0.006
5 -1 0 1 0.89 5.19 0.06 0.036 0.012
6 1 0 1 0.89 6.39 0.77 0.033 0.006
7 -1 0 -1 0.89 5.38 0.12 0.018 0.004
8 1 0 -1 0.88 5.64 0.06 0.022 0.006
9 0 -1 1 1.01 1.67 0.10 0.027 0.009
10 0 1 1 0.80 9.32 0.40 0.016 0.005
11 0 -1 -1 1.00 2.37 0.15 0.011 0.006
12 0 1 -1 0.81 6.17 0.37 0.013 0.007
13 0 0 0 0.89 5.29 0.04 0.029 0.006
14 0 0 0 0.89 5.58 0.04 0.032 0.007
15 0 0 0 0.89 5.86 0.18 0.025 0.004
The optimum conditions of extraction were determined by the technique of multi-response
analysis, following the algorithm proposed by DERRINGER and SUICH (1980). Each response is
transformed in a function denominated desirability function. The following global desirability
function was obtained:
Response surfaces obtained for the flavonoids content (TF) and solids content (Cs) as a
function of m
pl
/m
sol
and of the relation |m
w
/m
et
|, graphics (a) and (b) respectively.

The best condition of extraction were: temperature of 50
o
C, plant to solvent mass of 0.2,
the relation between the water to ethanol mass of 0.039.
( b) ( a )
The method of extraction selected in the preliminary tests was the heated stirred reactor.

m
m
m
m
96 . 0
m
m
38 . 0
m
m
86 . 0
m
m
77 . 2 62 . 5 TF
sol
pl
et
w
sol
pl
2
et
w
et
w
+ +
|
|
.
|

\
|
+ =
sol
pl
2
et
w
m
m
006 . 0
m
m
011 . 0 028 . 0 Cs +
|
|
.
|

\
|
=
(
(
(
(
(

(







|
|
|
|
|
.
|





\
|
+ + +
|
|
|
|
|
.
|





\
|
+
=
7 . 3
0 . 6
m
m
m
m
963 . 0
m
m
376 . 0
m
m
864 . 0
m
m
775 . 2 619 . 5
x
012 . 0
025 . 0
m
m
006 . 0
m
m
011 . 0 028 . 0
G
sol
pl
et
w
sol
pl
2
et
w
et
w
sol
pl
2
et
w