Open Journal of Forestry
2014. Vol.4, No.1, 24-27
Published Online January 2014 in SciRes (http://www.scirp.org/journal/ojf) http://dx.doi.org/10.4236/ojf.2014.41004
24
Isolation of Total Saponins from Sapindus mukorossi Gaerth
Menghao Du*, Sumei Huang, Jinping Zhang, Jingwen Wang, Lisong Hu, Jingmin Jiang
Research Institute of Subtropical Forestry, CAF, Fuyang, China
Received October 7th, 2013; revised November 16th, 2013; accepted November 30th, 2013
Copyright © 2014 Menghao Du et al. This is an o pen access article d istribu ted un der the Creative C ommons At-
tribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited. In accordance of the Creative Commons Attribution License all Copyrights ©
2014 are reserved for SCIRP an d th e o wner o f the in tellectual prop erty Menghao Du et al. All Copyright © 2014
are guarded by law and by SCIRP as a guardian.
The study has been carried out to investigate the effects of single factors such as solvents extraction tem-
perature, times, solid-liquid ration, and the time of extractions on the yields of saponins present in the
pulp of Sapindus mukorossi Gaerth. On this basis, an L9 orthogonal design of experiment was adopted to
determine the optimal conditions for the extraction of saponins. The factors that influence the extraction
of saponins are put in the order of extraction times, extraction time, solid-liquid ration, and the best com-
bination is that the powder of the pulp is extracted with EtOH (solid-to-solvent ratio = 1:8, w/v) for three
times at 60˚C for 3 hours. Under these conditions, about 1.63 g saponins will be extracted from 10 g raw
material. The stability test showed that the Sapindus mukuross saponins can maintain surface activity at
water temperature (25˚C - 40˚C), pH (6.3 - 7.7) and water hardness (50 - 250 mg∙L−1). It is proved that
Sapindus mukuross saponins are quality non-ionic active agent.
Keywords: Saponins; Sapindus mukorossi Gaerth; Orthogonal Design; Extraction
Introduction
The species of the genus of Sapindus belonging to the Sa-
pindaceae, has about five to twelve species of shrubs and small
trees (FRPS, 1998). Members of the genus are commonly
known as soapberries or soapnuts because the fruit pulp is used
to make soap (FRPS, 1998). There is about 10% saponins in
fruit pulp (FRPS, 1998), which makes it an ideal resource for
the extraction of Saponins. Saponin, a natural non-ionic surfac-
tant, not only has a good emulsifying, separating and dispersing
capability but also i s a good foam stabiliz er with a great clean-
ing capacity (Zhang et al., 1993). Thus, it can be used as foam
stabilizer for the building concrete (Lin, 1977), pesticide syn-
ergist (Hong & Tokunaga, 2000), and it also has antiviral, re-
ducing blood pressure functions (Huang et al., 2007; Ibrahim et
al., 2006, 2008; Kuo et al., 2005; Yukiyoshi, 2001; Yata et al.,
1986), so it is widely used in daily chemical industries, building
materials, food industries and agriculture.
Usually, saponin is extracted by using solvent like water,
ethanol and methanol (Huang et al., 2008; Rao & Sang, 2006).
Accordingly, this experiment will use ground pulp of Sapindus
mukorossi as raw material. Meanwhile the impact brought
about by different solvent, extraction times, extraction dura-
tions and different solid-liquid ratios has been investigated.
Materials and Methods
Plant and Chemical Materi a l
Fruits pulps of S. mukorossi were collected from Tiantai of
Zhejiang Province, China, in November 2008. Prior to all ex-
tractions, fruits pulp was dried at 60˚C for 48 h and was ground
in a Wiley mill to pass a 0.5 mm poresize screen. Chromato-
graph solvents used during the study were of HPLC grade and
the other solvents and reagents used during the study were of
AR grade.
Extraction Process
The main factors that affect the extraction of saponins like
extraction solvents, temperature, time, times and materi als ratio
(weight of the fruit pulp: volume of the extracting solvent),
were studied individually. The optimum extraction conditions
were determined by L9(34) orthogonal design of experiments i.e.
three levels and three different parameters.
Estimation of Total Saponins
Saponins sample was collected from extraction solution with
labware. Saponins concentration was measured in sample by
HPLC (column: SymmetryTM C18 (3.9 mm i.d × 150 mm),
40˚C, Flowing phase: CH3CN:H2O (H2O:90% → 20%, 30 min),
1ml∙mi n−1; detection wavelength: 210nm).
Stability Study
This study was carried out at vary water temperature (25, 35,
40˚C), pH (6.3, 7.0, 7. 7), water hardness (50, 100, 250 mg∙L−1)
with aqueous solutions having a saponin content of 4%. The
Cmc and γCmc were determined by dynamic tension meter.
Statistical Analysis
The results are expressed as means ± SD unless otherwise
*Corresponding author.

M. H. DU ET AL.
25
stated. The evaluation of statistical significance was determined
by the one-way ANOVA test, these analyses were done with
SPSS for WINDOWS, version 19.0, and with small letter <
0.05, capital letter < 0.01 considered to be statistically signifi-
cant.
Results and Discu ssion
Saponins in S. mukorossi Gaerth were extracted by nine sol-
vents, and its content was measured by HPLC. The best ex-
tracting solvents was ethanol which can result in the high
productivity of saponin, good quality of saponin colour and
volatiles (Table 1).
Figure 1 showed the yields of saponins tended to increase
with a rise in the temperature range from 20˚C to 50˚C. It may
be probable that the greater speed of the molecule movements
in higher temperature so that saponins diffused more quickly
from cell to extracting agent. But the yields of saponins could
be slight changed temperature of surpassing 60˚C. Tempera-
ture’s effect on extraction is dual. On one hand, higher temper-
ature can accelerate the solvent flow and thus increase the sa-
ponins content and on the other hand, higher temperature can
decrease the fluid density that may reduce the extraction effi-
ciency. Hence, it was found that 60˚C was the optimum tem-
perature for extracting the saponins.
Figure 2 showed the yields of saponins extracted was mini-
mum at 1:6 materials ratio. Further increase in the material ratio
leads to slight changed in the yields of saponins. This pheno-
menon might be due to the fact that when the material ratio
reached a certain level, the extract has well dissolved in the
solution that may lead the contents of the extract become satu-
rated and prevent further increase.
The yields of saponins extracted for 4 h reached maxima and
prolonged extraction may not increasing yield (Figure 3). T his
increase in the saponins content may be due to the synergistic
effect of other parameters involved.
The yields of saponins obviously increased with the no. of
extractions from 1 time to 2 times. But the yields of saponins
could be slight changed when the no. of extractions surpassing
2 times (Figure 4).
The parameters and the orthogonal design of experiment for
the extraction of saponins were given in the Tables 2 and 3.
bB
bB
bB
aA
bB
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
20℃40℃ 50℃60℃70℃
Extraction temperature
Yield of Saponins
(g/10g dry fruit pulp)
Figure 1.
Effects of temperature on the yield of S. mukorossi Gaerth. Saponins.
Ethanol, solid-liquid ratio 1:20, time 6 h, 2 extraction times.
Solid-solvent ratio
a
a
a
a
a
1.00
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.80
1：6 1：8 1：10 1：12 1：15
Yield of Saponins
(g/10g dry fruit pulp)
Figure 2.
Effects of the solid -solvent ratio on the yield of S. mukorossi Gaerth.
Saponins. Ethanol, temperature 60˚C, time 3 h, 2 extraction times.
1.00
1.10
1.20
1.30
1.40
1.50
1.60
0 1 2 345 6
a
b b b b b
Extraction time
yield of Saponins
(g/10g dry fruit pulp)
Figure 3.
Effects of extract time on the yield of S. mukorossi Gaerth. Saponins.
Ethanol, temperature 60˚C, solid-liquid ratio 1:10, 2 extraction times.
Table 1.
Effects of solvents on the characteristics of S. mukorossi Gaerth. Saponins (means ± SD).
Extracting solvents
MeOH EtOH Acetone Butanol H2O 95% MeOH 95% EtOH 95% Acetone 95% Butanol
Mass of
Saponins (g) 0.68 ± 0.12 1.54 ± 0.09 1.00 ± 0.12 0.98 ± 0.02 0.63 ± 0.11 1.19 ± 0.09 1.51 ± 0.06 1.37 ± 0.10 1.45 ± 0.07
Purity (%) 63.62 72.63 73.48 69.90 35.03 59.15 70.48 72.42 67.80
Desiccation
situation Easy Easy Easy Easy Difficult Little
viscosity Easy Easy Easy
Characters of the
dry substance yellow
powder
Off-white
powder
Light yellow
powder
Light yellow
powder
Brown
glue
Yellow
powder
Off-white
powder
Off-white
powder
Light yellow
powder
volatiles Slightly
sweet
special
fragrance
Special
fragrance
special
fragrance
Heavily
sweet
slightly
sweet
special
fragrance
special
fragrance
Special
fragrance
Note: Temperature 60˚C, solid-liquid r atio 1:20, time 6 h, 2 extraction times.

M. H. DU ET AL.
26
Table 2.
Factors and levels of orthogonal test.
Levels Extraction times (A) Solid-liquid ration (B) Extraction time (C)
1 1 1:8 2
2 2 1:9 3
3 3 1:10 4
Table 3.
Design and result of orthogonal test.
Test No. A B C Yield of S. mukor ossi Gaerth. Saponins (g)
1 1 1 1 1.05
2 1 2 2 1.34
3 1 3 3 0.92
4 2 1 2 1.48
5 2 2 3 0.89
6 2 3 1 1.29
7 3 1 3 1.54
8 3 2 1 1.57
9 3 3 2 1.61
k1 1.04 1.36 1.30
k2 1.22 1.20 1.41
k3 1.57 1.27 1.12
R 0.54 0.08 0.29
Table 4.
Influence of different factors on the surface activity of Sapindus mukuross saponins.
Temperature (˚C)
Water hardness
(mg∙L−1) pH
25 35 40 50 100 250 6.3 7.0 7.7
Cmc
(mg∙L
−1
)
32.9 24.8 21.6 39.3 56.8 76.9 33.4 60.4 76.8
γCmc (mN∙m−1) 36.3 35.2 34.2 35.9 36.1 37.4 36.5 37.6 38.2
b
b
b
a
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
1 23 4
Extraction times
Yield of Saponins
(g/10g dry fruit pulp)
Figure 4.
Effects of extraction times on the yield of S. mukorossi Gaerth. Sapo-
nins. Ethanol, temperature 60˚C, solid-liquid ratio 1:10, time 3 h.
The factors that influence the extraction of saponins are put in
the order of extraction times, extraction time, solid-liquid ratio,
and the best combination is when the powder of the pulp is
extracted with EtOH (solid-to-solvent ratio = 1:8, w/v) for three
times at 60 ˚C for 3 hours. Under these conditions, about 1.63 g
saponins will be extracted from 10 g raw material.
The results show that the Sapindus mukuross saponins can
maintain surface activity at water temperature (25˚C - 40˚C),
pH (6.3 - 7.7) and water hardness (50 - 250 mg∙L−1) (Table 4).
Conclusion
When the powder of the pulp was extracted with EtOH ( solid-
to-solvent ratio = 1:8, w/v) for three times at 60˚C for 3 hours,
namely in the best extraction condition, the largest yield of
saponins (1.63 g saponins will be extracted from 10 g raw ma-
terial) was obtained. The stability test showed that the Sapindus
mukorossi saponins can maintain surface activity at water con-
ditions under which people normally use detergent. It is proved

M. H. DU ET AL.
27
that Sapindus mukorossi saponins are quality non-ionic active
agent. Thus, we can conclude that this technology for saponins
extraction from S. mukorossi Gaerth. is efficient and environ-
mentally friendly.
Acknowledgements
The authors are grateful for the financial support by State
Forestry Administration P. R. China and Forest Department of
Zhejiang Province, China (Project number: 200804032,
2010SY03), for carrying out the above research work.
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