Isolation of Total Saponins from <i>Sapindus mukorossi</i> Gaerth

doi:10.4236/ojf.2014.41004

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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

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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

Email: *[email protected]

Received October 7th, 2013; revised November 16th, 2013; accepted November 30th, 2013

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 ©

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.

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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.

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

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

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.

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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

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.

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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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