Abstract: In Vietnam, Tam that hoang (Panax stipuleanatus Tsai et Feng) belonging to
Araliaceous family is a valuable medicinal plant that possesses many biologically active
compounds. Among them, flavonoids, polysaccharides, and saponins are the major
constituents of this plant. They have anti-oxidant, anti-microbial, anti-cancer, and tonic
properties. In this paper, we studied the factors that affect the process of total saponin
extraction from Tam that roots originated from Sapa town, Lao Cai province. The total
saponin extraction from Tam that roots was optimized using the desirability methodology
with the experimental order of Box-Behnken design. The concentrations of ethanol, the ratio
of ethanol/material (v/m), the time of ultrasonic extraction with the frequency of 33 - 40 kHz
are the three main considered factors. The optimized condition for total saponin extraction
was found as follows: ethanol concentration 65%, ethanol/material ratio 55/1(v/m), and
extraction time 75 minutes. In this condition, the maximum total saponin content was
predicted to be 15.01%.
Keywords: Extraction, saponin, Pan

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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0052
Natural Sci. 2016, Vol. 61, No. 9, pp. 26-33
This paper is available online at
26
OPTIMIZATION OF TOTAL SAPONIN EXTRACTION FROM TAM THAT ROOTS
USING THE DESIRABLITY METHODOLOGY
Nguyen Quang Tuyen and Dang Ngoc Quang
Faculty of Chemistry, Hanoi National University of Education
Abstract: In Vietnam, Tam that hoang (Panax stipuleanatus Tsai et Feng) belonging to
Araliaceous family is a valuable medicinal plant that possesses many biologically active
compounds. Among them, flavonoids, polysaccharides, and saponins are the major
constituents of this plant. They have anti-oxidant, anti-microbial, anti-cancer, and tonic
properties. In this paper, we studied the factors that affect the process of total saponin
extraction from Tam that roots originated from Sapa town, Lao Cai province. The total
saponin extraction from Tam that roots was optimized using the desirability methodology
with the experimental order of Box-Behnken design. The concentrations of ethanol, the ratio
of ethanol/material (v/m), the time of ultrasonic extraction with the frequency of 33 - 40 kHz
are the three main considered factors. The optimized condition for total saponin extraction
was found as follows: ethanol concentration 65%, ethanol/material ratio 55/1(v/m), and
extraction time 75 minutes. In this condition, the maximum total saponin content was
predicted to be 15.01%.
Keywords: Extraction, saponin, Panax stipuleanatus.
1. Introduction
Extraction is the first step of great importance for the recovery and purification of active
ingredients from plant materials. Traditional extraction techniques usually require many extraction
hours and have low efficiency. Recently, there have been numerous reports on the application of
high intensity or power ultrasound in the extraction of various phyto-chemicals such as alkaloids,
flavonoids, poly-saccharides, proteins and essential oils from various parts of plants and their seeds [1-3].
The ultrasound-assisted extraction can be done at a low temperature to avoid the decomposition of
natural compounds and essential oil. Even so there are several advanced extraction technologies,
ultrasound-assisted extraction using ethanol solvent is a reasonable choice, since it is economical
and can be applied in industry without toxic residues [4-7].
Statistical experimental designs are proved to be more satisfactory and effective than other
traditional which only allow us to study one-at-a-time factor or mathematical methods because it
can be used to study many variables simultaneously, with less observation. This method has been
widely used in different research fields such as biology, agriculture, food, and medicine etc.
Received October 10, 2016. Accepted November 25, 2016.
Contact Dang Ngoc Quang, e-mail address: quangdn@hnue.edu.vn
Optimization of total saponin extraction from Tam that roots using the desirablity methodology
27
Desirability methodology is considered one of the effective methods for multiple objective
optimizations of technical factors [8]. In this study, the desirability methodology was employed to
optimize total saponin extraction from dried powder of the roots of Panax stipuleanatus using
ethanol as solvent.
2. Content
2.1. Experiment
2.1.1. Material
Roots of Panax stipuleanatus, were collected in Sapa town, Lao Cai province, northern part
of Vietnam in May 2013, with samples identified by Mrs. Pham Thanh Huyen from National
Institute of Medicinal Materials. Voucher specimens were deposited at the Faculty of Chemistry,
Hanoi National University of Education (N
0
-9754; 9755). Materials were collected, cleaned
steamed at 100
0
C for 5 minutes, and sliced, then dried at 60
0
C for 5 days to the moisture of 5 - 6%,
finally powdered with the size of particles of 0.1 - 1 mm. Dried samples were kept in two-layer
polyethylene bags, placed into glass bottles and stored in a dry and cool place.
2.1.2. Analysis methods
Quantitative analysis of the total saponin was carried out using the analytical balance. In
addition, the saponin content was determined by HPLC (Hitachi) with standard saponins
(notoginsenosid R1, ginsenosid Rg1 and ginsenosid Rb1), equipped with a reversed phase column,
phenomenex C18 (5 m × 250 mm × 4.6 mm) using acetonitrile and water as a solvent system.
2.1.3. Research methods
Extraction: Dried powder of Tam that roots (approximately 2 g) was placed in a 250 mL
flask. The extraction was carried out in a thermo-regulatory ultrasound machine with an ethanol
concentration 50 - 90%, ethanol/material ratio (30 - 60/1, v/m), and extraction time 30 - 90
minutes. The extract was filtered and solvents were removed by a rotary evaporator. The residue
was finally dried by a vacuum dryer. The total saponin was calculated by a percentage of the
sample (% dry substance).
Statistical experimental design method: Using the statistical Box-Behnken design with three
factors: ethanol concentration (%), ethanol/material ratio (v/m) and ultrasonic extraction times
(minutes), (Table 1). This design has 15 trials including 3 ones for center points to response: total
saponin (% dry substance), (Table 2).
Table 1. The factors and their levels
Factors Symbols Units
Levels
-1 0 +1
Ethanol
concentration
A % 50 70 90
Ethanol/material B v/m 30 45 60
Ultrasonic times C minutes 30 60 90
Nguyen Quang Tuyen and Dang Ngoc Quang
28
Table 2. Experimental design and total saponin
Run
Ethanol
concentration
(%)
Ethanol/material
(v/m)
Ultrasonic
times (minutes)
Total saponin
(%)
1 50.00 30.00 60.00 9.65
2 90.00 30.00 60.00 6.49
3 50.00 60.00 60.00 12.35
4 90.00 60.00 60.00 9.16
5 50.00 45.00 30.00 9.99
6 90.00 45.00 30.00 7.04
7 50.00 45.00 90.00 11.98
8 90.00 45.00 90.00 8.56
9 70.00 30.00 30.00 9.97
10 70.00 60.00 30.00 10.79
11 70.00 30.00 90.00 9.85
12 70.00 60.00 90.00 14.41
13 70.00 45.00 60.00 14.17
14 70.00 45.00 60.00 14.16
15 70.00 45.00 60.00 14.18
2.1.4. Statistical analysis
The statistical Design-Expert 7.1 software (Stat-Ease, Inc., Minneapolis, USA) was used for
regression analysis of experimental data to plot response surface and to optimize, according to the
desirability methodology [9]. ANOVA (Analysis of variance) was used to estimate the
statistical parameters.
2.2. Results and discussion
2.2.1. Study on the influence factors on total saponin extraction
The total saponin amount depended on many factors such as ethanol concentration (%)
(Figure 1), ethanol/material ratio, (v/m) (Figure 2) and ultrasonic times (minutes) (Figure 3) [10, 11].
In our work, each single factor was studied in order to find out its effect on total saponin
extraction. Based on those, we will choose a value range of each factor for optimization. For
example, the ultrasonic time (60 min) and the ethanol/material ratio (v/m: 50/1) were fixed, but
ethanol concentration was varied to find out the value range of ethanol concentration.
Optimization of total saponin extraction from Tam that roots using the desirablity methodology
29
Figure 1. Ethanol concentration (%)
Figure 2. Ethanol/material ratio (v/m)
Figures 1 - 3 show that total saponin extraction depended on all three 3 factors. Based on the
experimental data, we decided to choose value ranges of each factor as follow: ethanol
concentration 50 - 90%, ethanol/material ratio, 30/1 - 60/1 (v/m), and time of extraction using
ultrasound 30 - 90 min.
Figure 3. Time of extraction using ultrasound (min)
2.2.2. Model building
The results of experimental design are shown in table 2. The response total saponin was fitted
with a second-order polynomial equation of the three factors. The significance of the model and
the co-efficients are controlled by a regression analysis (Table 3). The F-values of the model are
1.916.10
5
showing that the model is completely significant of 99.99% (p < 0.0001) confidence
level. The p-value less than 0.05 indicating that the coefficient is significant, therefore, in the
model only interactive coefficient AB (p = 0.099) was insignificant, but it was remained in the
model for optimization. F-value for “lack of fit” of the model was 0.25 (p = 0.8576). It showed
that the model was fit with the experiment. In addition, the coefficient of determination (R
2
) of the
model 0.999 showed that the model was well-matched with the designed experiment. The total
saponin is performed in a second-degree model as follows:
Y = 14.17 - 1.59A + 1.34B + 0.88C - 7.5.10
-3
AB - 0.12AC + 0.94BC - 3.31A
2
- 1.45B
2
- 1.47C
2
Nguyen Quang Tuyen and Dang Ngoc Quang
30
Table 3. Regression analysis of total saponin
Source Mean square F-value
p-value
(prob > F)
Model
A
B
C
AB
AC
BC
A2
B2
C2
Lack of fit
94.85
20.22
14.45
6.14
2.250E-004
0.055
3.50
40.45
7.74
7.95
2.00E-005
1.916E + 005
3.677E + 005
2.626E + 005
1.117E + 005
4.09
1004.09
63580.00
7.355E + 005
1.407E + 005
1.446E + 005
0.25
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.0990
< 0.0001
< 0.0001
< 0.0001
< 0.0001
< 0.0001
0.8576
Considering in turn the effect of each factor on the total saponin extract (while other factors
are remained in the central level) (Figure 4), it showed that the ethanol concentration (A) has
played a significant role on the total saponin content: meanwhile ethanol/material ratio (B) and
ultrasonic time (C, time of extraction) took the same role on the total saponin. This can be shown
obviously in the response surface of Y-function (Figures 5a, 5b, 5c).
Figure 4. Influence of factors on total saponin extraction
Optimization of total saponin extraction from Tam that roots using the desirablity methodology
31
5a
5b
5c
Figure 5. Response surface plot for total saponin extraction
2.2.3. Optimization of the total saponin extraction
The most important objective of total saponin extraction process from Tam that roots is to get
the highest total saponin. To this point, the optimization using algorism of desirability
methodology invented by Derringer and Suich was applied [8]. The results after using the Design-
Expert 7.1 Software were as follows: ethanol concentration 65%, ethanol/material ratio 55/1
(v/m), and time of extraction using ultrasound 75 min. In this condition, the predicted maximum
total saponin extract was 15.01% (Figure 6).
Figure 6. Responsible desirability level
Nguyen Quang Tuyen and Dang Ngoc Quang
32
2.2.4. Validation of the model
The trial experiments were conducted (four times) under optimized process conditions with
ethanol concentration 65%, ethanol/material ratio 55/1 (v/m), and time of extraction using
ultrasound 75 min. The mean value of the total saponin has reached 15.00 ± 0.02 % (Table 4),
which was very close to the predicted total saponin obtained from the regression equation (15.01 %).
Thus, the model could be used to predict the total saponin content obtained from the extraction.
Table 4. The extraction results under optimal condiction
Number
Ethanol
concentration
(%)
Ethanol/material
ratio
(v/m)
Time of
extraction
(min)
Total
saponin
(%)
1 65 55/1 75 15.01
2 65 55/1 75 14.99
3 65 55/1 75 15.01
4 65 55/1 75 14.98
Average 15.00 ± 0.02
3. Conclusion
Based on the statistical experimental design using response surface and desirability
methodology, the optimal condition for total saponin extraction by ethanol solvent was
determined as follows: ethanol concentration 65%, ethanol/material ratio 55/1 (v/m), and time of
extraction using ultrasound 75 min. The maximum total saponin content was predicted to be 15.01%.
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