CHAPTER IV RESULT AND DISCUSSION
4.1. The investigation of saponin content extracted from Sapindus rarak DC . 45
process in various condition was conducted. The extraction process was performed by two different methods for various extraction condition. The first method was conventional water maceration extraction (ME) and the green ultrasound-assisted extraction (UAE) methods. The maceration extraction is a solid–liquid extraction where the bioactive compound (solute) inside the plant material is extracted by soaking the plant material in a specific solvent for a period of time (Takeuchi et al., 2009). The efficacy of maceration process is determined by two main factors, solubility and effective diffusion. The solubility is governed by basic rule of “like dissolves like” which indicated that polar compounds dissolve in polar solvents, and nonpolar compounds dissolve in nonpolar solvents (Reichardt and Welton, 2011). In the other hand, UAE methods utilize the ultrasonic wave to help the extraction process. The phenomenon of ultrasound in creating cavitation bubbles in the solvent by acting as a micro jet to denature the plant cell wall when the bubbles collapse at rare fraction resulted in a greater extraction yield of bioactive compounds. Few researchers have reviewed the applications of ultrasound-assisted extraction on bioactive principles from herbs (Vinatoru, 2001), food industry and
46 processing (Vilkhu et al., 2008). Figure 4.1 present the yield of saponin at various the extraction time for both UAE and ME methods.
Figure 4.1 Effects of extraction time on the saponin content of the Sapindus rarak extracts, at temperature of 30oC and solid-liquid ratio of 2 mL/gram
The UAE reaches the maximum saponin content at the time of 40 minutes at total saponin content of 18.708 mg/100 mg feed. On the other hand, saponin content of 15.186 mg/100 mg feed was obtained for ME at the extraction time of 120 minutes, which is lower than those of the UAE. According to Figure 4.1, the effect of extraction time was different on the UAE and ME. On both process, it can be seen that the saponin yield was increased by the extraction times until a specific time. And then the yields were constant or slightly decreased by the extension of processing time. The results indicates that with or without ultrasound irradiation, the diffusion of the bioactive compounds from material to solvent tend to increase by the time and the equilibrium for dissolution is established after a certain time.
The increase of saponin yield by the time extension indicates that the solubilization of saponin compound to the solvent requires a certain time. The extraction with assistance of ultrasonic waves reach its highest yield at a shorter time than the maceration. The assistance of ultrasonic waves helps to release the saponin compound into the solvent by destructs the plant tissues. This process allowing more saponin to treanfer into the solvent in shorter time.
Extraction Time (minutes)
0 20 40 60 80 100 120 140
Saponin Yield (mg/100mg feed)
10 12 14 16 18 20
UAE ME
47 However, it is possible that the components degrade after a long exposure of ultrasonic waves. The decline of saponin content at the end of the UAE indicates that an excess extraction time is not preferable. Ultrasonic wave has an ability to disturb a plant material. At the beginning of the process this disruption is desired.
However, a longer exposure to ultrasonic wave can also disturb the desired bioactive compound, saponin, which lowering the saponin yield. The similar results were also reported on the previous study about UAE of anthocyanin and phenolic from purple sweet potato (Zu et al., 2016), extraction of natural antioxidant from Jatropha integerrima (Xu, et al., 2015) and Boldo leaves (Petigny et al., 2013).
Tarade et al. (2006) reports the degradation phenomena of saponin from soybean flour in the food processing by slow cooking, and long heating. As much of 81-84%
of saponins were reduced in the soaked, dehulled and autoclaves sample of faba beans (Sharma and Sehgal, 1992). The degradation of saponin also found in the extraction of chickpeasaponin by microwave assisted extraction (Cheng et al., 2017). The study by Cheng et al. (2017) also reports that the reduction of saponin contents depends on the processing parameters such as solid/liquid ratio, extraction temperature, microwave irradiation power, irradiation time, and pH.
Figure 4.2 The effect of solvent to material ratio on the saponin content of the Sapindus rarak extracts at 30oC for 120 minutes
Figure 4.2 presents the effect of various ratio of solvent to the dried-ground Sapindus rarak to the yield of saponin extracted. The yield of saponin was
Ratio of Solvent to Feed (mL/gr)
0 20 40 60 80 100
Saponin Yield (mg/100 mg feed)
10 12 14 16 18 20 22 24 26 28 30
UAE ME
48 calculated on dry based. The saponin content (ppm) found from the spectroscopy analysis was recalculated to obtain the content in milligrams of saponin per mL of total solvent. The total extracted saponin in a batch of solvent (milligrams) was then divided by the amount of total dry feed in the same batch of solvent (milligrams).
The study was conducted for both UAE and ME process. From the previous investigation, since the maximum yield was obtained after 40 minutes of extraction, then the extraction were conducted for 40 minutes, at the extraction temperature of 30 oC. Based on Figure 4.2, it can beSSS seen that the saponin content increase by the addition of solvent and then slightly decrease or stay stable with a further increase of solvent. The maximum saponin yields obtained from the UAE process was 27.871 mg/100 mg feed at the addition of 10 mL of solvent per gram of solid component. While in the ME process, the saponin content remain constant at the addition of 50 mL of solvent per gram solid, with the maximum saponin yield achieve was only 20.586 mg/100 mg feed.
The extraction process of saponin from Sapindus rarak was conducted based on the solubility of saponin on to the solvent. Saponin is an amphiphilic molecule having ability to solubilize well on both water or oil/fat soluble solvent. However, there is a possibility of fatty component to be solubilize together with the saponin if a fat soluble solvent is used (Hierro et al., 2018). The existence of fat in the solution is going to disturb the saponin performance as a surfactant (Reichert et al., 2017; Li et al., 2013). The separation of fat from the crude saponin solution difficult and complicated, therefore, water was chosen as the solvent to extracted saponin.
With the used of water as the solvent, the saponin will be solubilized with the addition of solvent. By increasing the volume of solvent, the dissolved materials is higher and resulted in more extraction yield. In addition, the mass transfer parameter is affected by the amount of solvent volume. More solvent results on enlargement of mass transfer parameter and accelerate the diffusion of molecule into the medium (solvent) (Xu et al., 2016). The solvent also have an effect to increase the diffusion of the component to the solvent, resulting to the dependence of extraction rate on the particle concentration gradient. Furthermore, the extraction is influenced by how fast the component is dissolved and the equilibrium in the
49 liquid achieved (Cacace, 2003). However, at a point, the further addition of solvent gives no difference due to the saturation of solvent by the extracted compound.
After saturation point, the solvent cannot contain any further component. The extraction of other natural compound such as anthocyanin and phenolic components of black currants also shows a similar phenomenon. The amount of extracted anthocyanin increased as the solute-solvent ratio increases until specific points (Cacace, 2002). Similar results were also found in anthocyanin extraction by UAE from Mulberry (Zou et al., 2011), extraction of antioxidant from the flower of Jatropha integerrima (Xu et al., 2016), extraction of phenolics from wine lees (Tao et al., 2014) as well as ursolic acid extraction from Ocimum sanctum leaves (Vetal et al., 2012).
Figure 4.3 The effect of extraction temperature on the saponin content of the Sapindus rarak extracts at solvent to material ratio of 10 mL/g, for 40 min
The effect of temperature on to the saponin content in the extract solution is presented in Figure 4.3. The Figure indicates that increase of temperature condition has different effect for different extraction methods. The extraction by maceration process shows a positive dependency of extracted saponin with the increase of temperature. The saponin concentrations increase as the temperature rises. The highest saponin yield of 19.888 mg/100mg feed was obtained at the extraction temperature of 50 oC. And the increase of temperature to 60 oC reduced the saponin yield to 18.842 mg/100mg feed, meaning that in 10 mL of solvent, a gram of Sapindus rarak powder can produce 188.42 mg of saponin. Considering that the
Extraction Temperature (oC)
30 40 50 60
Saponin yield (mg/100 mg feed)
0 5 10 15 20 25 30
UAE ME
50 maximum solubility of saponin in water is 50 mg/mL (Sigma-Aldrich, Singapore), the maximum amount of soluble saponin in the 10 mL of water solvent is 500 mg.
Shows that the extraction of saponin in 10 mL of water was considered possible and the reduction of saponin yield was not caused by the solvent saturation. A slight decrease of saponin yield by the addition of heat was due to the heat degradation of saponin. Bioactive compound in liquid extract form was known to be more susceptible to heat (Peron et al., 2017). Bioactive compound such as anthocyanin shows a degradation at the temperature of 50-60 0C (Aurelio et al., 2008).
While, the extraction process with the assistance of ultrasonic wave shows a different phenomena the rise of temperature decrease the saponin content. The highest saponin yield of 27.871 mg/100mg feed was obtained at the temperature of 30 oC. The addition of heat decreased the yield to 22.551 mg/100mg feed at extraction temperature of 60oC. A significant decline of saponin yield was found in the extraction with assistance of ultrasonic waves. This phenomena shows that a long exposure of ultrasonic waves on higher temperature condition was able to gaves destructive effect to the saponin compound, in which, decrease the saponin yield significantly. In the UAE process, the exposure of ultrasonic waves producing a lot of cavitating bubbles which creates local increase of temperature in the feed surface called hotspot. The hotspot induced a destructive effect which helps the extraction process under right condition (Alupului et al., 2009).
Comparison of UAE and ME reveals that the UAE gives a better result. Figure 4.1 shows that UAE process was faster and gaves higher saponin content compared to ME. The maximum yield of the saponin obtained from the ME process not even surpassed the saponin yield obtained from the first 5 minutes of UAE. Figure 4.2 shows that UAE requires less solvent to extract saponin compares to ME. By using ultrasonic wave in the extraction process, about 80% of required solvent can be reduced. While, Figure 4.3 showing that UAE need lower heat to extract saponin compared to ME. Even the addition of temperature can increase the saponin content in the ME process, the maximum yield cannot compared the yield achieved by UAE process. The UAE process achieved its maximum yield on 30oC, while the ME
51 achieved its maximum yield on 50oC. The extraction mechanism of saponin through the ME and UAE was illustrated in Figure 4.4.
Figure 4.4 Schematic illustration of (a) Saponin in plant tissue, (b) ME mechanism and (c) UAE mechanism
Considering the research result thoroughly, the saponin for the further used in this study will be extracted using UAE process on its optimum condition where the highest saponin yield obtained. The detail of maximum condition of each process (UAE and ME) was presented in the Table 4.1.
Table 4.1 The optimized parameter of the saponin extraction by UAE and ME
Extraction Methods
Time (minutes)
Ratio of solvent to solid
(ml/gr)
Temperature (oC)
Maximum saponin yield (mg/100mg
feed)
UAE 40 10 30 27.87125
ME 120 50 50 23.7365
(a)
(b) (c)
52 4.2. Characterization of saponin as surfactant
The extract with highest saponin yields used for the further analysis. The concentration of saponin in the extract was also known, and used as the calculation basis (CMC, molar). The saponin characterization was conducted by the investigation of functional group using FTIR, the analysis of surfactant CMC, foaming properties analysis and the value of hydrophilic and lipophilic balance (HLB). All of the investigation was conducted with comparison to the commercialized pure saponin.
4.2.1 Specific Functional Group
Figure 4.5 shows the FTIR spectra of extract saponin from Sapindus rarak DC and commercialized pure saponin. In general the peak of sapoin extract was similar with the peak of the pure saponin. Almost all of the relative peaks shows on the pure saponin FTIR was also showed in the saponin extract. This confirms that the extract of Sapindus rarak contain saponin. Both samples shows the infrared absorbance characteristic of the hydroxyl group (OH) at 3393.90 cm-1 in the pure saponin and at 3421.77 cm-1 in the saponin extract. Carbon – hydrogen (C-H) absorption at 2932.16 in the pure saponin and at 2934.24 cm-1 in the saponin extract.
The C=C absorbance was observed at 1609.73 cm-1 and 1638.54 cm-1 in the pure saponin and saponin extract respectively. The peak of oligosaccharide linkage which indicating the existence of sapogenins (aglycone), C-O-C, were also present at 1077.05 cm-1 in the pure saponin and at 1047.13 cm-1 in the saponin extract. The peak at 1412.58 cm-1 in pure saponin and 1388.07 cm-1 in saponin exract indicating the group of –CHO from the oleanane structure. A weak peak at 1243.9 cm-1 in pure saponin and 1261.12 cm-1 in saponin extract indicating the aromatic =C-H and a broad peak at 617.7 cm-1 and 656.48 cm-1 in pure saponin and saponin extract presents the pyranose sugar at the glycone structure. The presence of both water soluble glycone structure and the hydrophobic aglycone structure in the FTIR spectrum shows that the sample has the saponin characteristic. The IR spectrum of extract Sapindus rarak also shows a high similarity to the pure commercial saponin, proving that the extracts contains saponin.
53 Figure 4.5 The FTIR spectra of pure saponin and extract of saponin from Sapindus rarak
DC.
The aforementioned characterization of saponin utilize the functional group infrared absorptions has been conducted for soapnuts, quilaja saponaria (Almutairi and Ali, 2014), basellasaponin (Toshiyuki et al., 2001) and steroidal saponin (Da silva et al., 2002). The previous study reports that the existence of –OH, C-H, C=C, and C-O-C were identified as oleanane triterpenoid saponins (Kareru et al., 2008).
The study also states that the C=O groups can be either exist or not. The present of C=O groups indicates a bidesmosidic saponin (Kirmizigul et al., 2002). However, in Figure 4.5 there is no infrared absorption to confirm the present of C=O groups.
Hence, the saponin was presumably a monodesmosidic oleanane triterpenoid saponins. The peak of C=O group also cannot be found for the pure commercial saponin. Indicating that the pure commercial saponin used in this study has a good compatibility with the saponin from Sapindus rarak. The molecular structure of saponin from sapindus rarak has also been provided by Morikawa et al. (2009), which is presented in Figure 4.6. The observation of saponin molecule from various plant has also been conduted by Kareru et al. (2008) and showing a similar results.
54 Figure 4.6 Molecular structure of Sapindus rarak (monodesmosidic oleanane
triterpenoid saponin)
4.2.2 Critical Micelle Concentration
The saponin characterization as a surfactant was also conducted to find the critical micelle concentration (CMC). A knowledge of the critical micelle concentration (CMC) of the extract of saponin from the plant material is very important in the context of its applications. The data was required in the surfactant- integrated process such as foaming, emulsion stabilisation, as well as the incorporation of lipophilic compounds into micelles, as in micellar-enhanced ultrafiltration (MEUF). At the air-water or oil-water interface the hydrophilic side chains of saponins are directed towards the aqueous phase. The hydrophobic aglycone faces towards the nonpolar air or oil phase. This results in a decrease of the water’s surface tension or the interfacial tension between water and oil, respectively, which proceeds until the CMC is reached. At the CMC the interface is saturated with surfactant molecules. Further increase of the saponin content leads to self-aggregation of the surfactant molecules in the aqueous phase, resulting in the formation of micelles. Thus, no further decrease in surface (or interfacial)
55 tension can be observed. The main reason for micelle formation is the attainment of a minimum free energy state. The illustration of micelles formation in water solution is presented in Figure 4.7
Figure 4.7 The micelle formation in water solution, (A) Below CMC, (B) Right at CMC, and (C) Above CMC
The CMC was determined by the surface tension decline for both of pure saponin and extract of saponin from Sapindus rarak. The surface tension of water at various concentration of saponin is present in Figure 4.8.
(a) (b)
Figure 4.8 Surface tension of (a) Saponin extract in aqueous solution, (b) Pure saponin in aqueous solution, at various concentration
To determine the CMC of the saponin extract, the measurement of surface or interfacial tension of aqueous saponin solutions in various concentrations was conducted. The surface tension was declined with the addition of saponin for both
Concentration of Saponin Extract (%)
0 1 2 3 4 5 6 7 8 9 10
Surface Tension (dyne.cm-1) 40 50 60
70 Saponin Extract
CMC (%)
Concentration of Pure Saponin (%)
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Surface Tension (dyne.cm-1) 35 40 45 50 55 60 65 70 75
Pure Saponin
CMC (%)
56 extract saponin and pure saponin solution as clearly presented in Figure 4.8. At a certain point the surface tension stops decreasing with a further addition of saponin and remain almost constant. The surfactant concentration at which the surface tension reached its constant value considered as the CMC of saponin. The constant value was taken at the point where three or more point after them is remain the same. The critical micellar concentration was found to be 0.07 wt % saponin for pure saponin and 7 wt % of saponin extract for aqueous saponin extract solution.
The CMC of pure saponin is corresponds with the CMC provides by the manufacturer, which is 0.001-0.1% wt.
There is a huge difference between the CMC of pure saponin and saponin extract. The value of 7% of weight was calculated based on the addition of aqueous S. rarak extract to the solution. However, if the calculation was conducted based on the saponin content in the solution, the CMC was found to be 0.19% wt. The value was higher than the calculated CMC of the pure saponin. The aqueous rarak extract was not purified completely like the commercial one. Even after simple purification using centrifugation, a little amount of impurities such as starch was supposed to remain in the solution. The starch in the solution can disturb the molecules of surfactant to assembly in the solution surface. The solution of saponin extracts were also thicker than the pure saponin solution, indicating a more viscous solution. In viscous solution, the surface tension was tend to be a little bit higher. The addition of surfactant to lowering the surface tension was then interrupted by this properties.
Resulting to a higher CMC of the extract solution compared to the pure saponin.
The similar results also found in the previous study on the surfactant production from the pericarps of Sapindus muccorosi, where the CMC of the crude solution was higher than the standard saponin (Samal et al., 2017). If the CMC expresses in millimolar (mM), the CMC of saponin extract and the pure saponin are 3.075 mM and 1.102 mM respectively.
4.2.3 Foam Ability of Saponin
Liquid films are the basic structural elements of any foaming system containing surfactant micelles. Consequently, the stability of the foam is highly dependent on