Isolation of Total Saponins from Sapindus mukorossi Gaerth

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Introduction
The species of the genus of Sapindus belonging to the Sapindaceae, 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 surfactant, not only has a good emulsifying, separating and dispersing capability but also is a good foam stabilizer with a great cleaning capacity (Zhang et al., 1993). Thus, it can be used as foam stabilizer for the building concrete (Lin, 1977), pesticide synergist (Hong & Tokunaga, 2000), and it also has antiviral, reducing blood pressure functions (Huang et al., 2007;Ibrahim et al., 2006Ibrahim et al., , 2008Kuo 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 durations and different solid-liquid ratios has been investigated.

Plant and Chemical Material
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. Chromatograph 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 materials ratio (weight of the fruit pulp: volume of the extracting solvent), were studied individually. The optimum extraction conditions were determined by L 9 (3 4 ) orthogonal design of experiments i.e. three levels and three different parameters.

Statistical Analysis
The results are expressed as means ± SD unless otherwise 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 significant.

Results and Discussion
Saponins in S. mukorossi Gaerth were extracted by nine solvents, and its content was measured by HPLC. The best extracting 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. Temperature's effect on extraction is dual. On one hand, higher temperature can accelerate the solvent flow and thus increase the saponins content and on the other hand, higher temperature can decrease the fluid density that may reduce the extraction efficiency. Hence, it was found that 60˚C was the optimum temperature for extracting the saponins. Figure 2 showed the yields of saponins extracted was minimum at 1:6 materials ratio. Further increase in the material ratio leads to slight changed in the yields of saponins. This phenomenon 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 saturated and prevent further increase.
The yields of saponins extracted for 4 h reached maxima and prolonged extraction may not increasing yield (Figure 3). This 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.   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.   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.

Conclusion
When the powder of the pulp was extracted with EtOH (solidto-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 material) was obtained. The stability test showed that the Sapindus mukorossi saponins can maintain surface activity at water conditions under which people normally use detergent. It is proved 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 environmentally friendly.