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A Study of the Extraction of Solasodine from Solanum Hainanense
Methods
1. Determination of some main components in Solanum hainanense Hance
- Determination of the average moisture content of the original material: drying 10g sample at 1050C to constant weight.
- Determination of ash content: drying 10g samples at 1050C for 2 hours, then heating at 6000C to constant weight.
- Quantification of reducing sugars according to Bertrand method.
- Quantification of starch by acid hydrolysis method - Quantification of cellulose - Protein quantification by Kjeldahl method.
2. Survey the effects of drying method on the extraction process
Weighing 6 fresh leaf samples, each of 20 g, then drying 3 samples by convection drying method and drying 3 samples by vacuum drying method for 5 hours at 500C.
3. Survey the drying temperature of raw materials affecting the extraction process Drying at different temperature conditions affected the content of soladine compounds present in Solanum hainanense Hance. We carried out the drying process at 500C, 550C, 600C for 5 hours, by vacuum drying method.
4. Survey the drying time affecting the extraction process
The drying time of the material ensured that the moisture content of the products reached the moisture required for storage and increased the extraction efficiency.
We investigated drying time of 5 hours, 6 hours, 7 hours, 8 hours. 12 fresh samples were weighed at 20g, then 3 samples were dried from 5 hours to 8 hours, at 50oC, by vacuum drying.
In items of 2.2.2, 2.2.3 and 2.2.4, to evaluate the effectiveness of the methodological investigations, the temperature and drying time influences the extraction process. We used the method measuring optical density UV-VIS at wavelength 206nm to select the optimum elements during drying. Weighing each sample 5g into a Erlenmeyer flask at 50° C with 95% ethanol, material /Solvent = 1/10 for 3 hours. After extracting, the filter was taken to extract the extraction and then diluted to measure OD.
5. Survey the effect of solvent on solasodine extraction from Solanum procumbens To investigate the effect of solvents on solasodine extraction, we used three solvents: 95%
ethanol, acetone, distilled water, and three chemicals with increased polarization as solvents for solasodine extraction from Solanum procumbens.
6. Survey the effects of the material:solvent ratio analysis (Solids:Solvent) on the extraction process
We conducted the following material / solvent ratios: 1: 4 (S1), 1: 5 (S2), 1: 6 (S3), 1: 7 (S4) 1:10 (S5), 1:10 (S7), 1:15 (S8) corresponding to the amount of solvent (20, 25, 30, 35, 40, 45, 50 and 75ml) for 5g samples.
7. Survey the effects of temperature on the extraction process
In this study, we used a 95% ethanol solvent with a material / solvent ratio of 1/10 (g /ml) for 3 hours, surveying at 5 different temperature levels: 400C, 450C, 500C, 550C, 600C.
8. Survey the effects of extracting time on the extraction process
We conducted the survey during 3 hours, 4 hours and 5 hours at 550C, using ethanol as a solvent with M / S ratio: 1/10, then filtrating and measuring OD206nm.
9. Qualitation of solasodine by HPLC Chromatography
The solasodine extract of Solanum hainanense Hance was filtered with a 0.2 μm Minisart membrane (Sartorius, Germany), diluted 5 times in the same solvent and stored at 4 ° C.
Condition:
- Hypersil MOS (C8) (5 μm, 4.6 × 150 mm) column of Thermo Scientific - Runtime: 5 minutes / sample
- Flow rate: 1 mL / min
The detector has a wavelength of 254 nm.
- Static phase is silicagel (back phase) - Mobile phase is 100% methanol.
HPLC analysis was conducted on a Shimadzu LC 20A system with LC-20AD pumps, SPD-20A probes, SIL-SPD-20A HT samplers and LC Solution software (ver.1.22).
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10. Experimental method for investigating the formula of beverage from Solanum hainanense Hance
- Survey the concentration of solasodine solution content in sugar-free Solanum hainanense Hance water-based product formulation: solid content of solasodine was calculated by volume in 100 ml of water in the following proportions: 0.25 ml; 0.5ml; 0.75ml; 1ml; 1,25ml;
1.5ml. After mixing, the formula of drinks was evaluated by sensory evaluation method, using a 9-point Hedonic Scale.
- Survey the saccharose and citric acid contents in formulations containing sugar: Sugar was added to a 70% syrup formulation at 100 volume ratios of water at 1%, 2%, 3%, 4%, 5%, 6%.
At the same time, citric acid was added to the formula at a volume ratio of 0.01%; 0.02%; 0.03%, 0.04%. In it, the solsodine solution is added at 1 ml in 100 ml of water. After mixing, the formula of drinks was evaluated by sensory evaluation method, using a 9-point Hedonic Scale.
- Survey the isomalt sugar content in drink from Solanum hainanense Hance: Isomalt added solids dissolved in water and adjusted for 100% water content in percentages: 5%, 10%, 15%, 20%, 25%, 30%. In it, the solid solsodine solution is added at 1 ml in 100 ml of water. After dosing, the formula of drinks was evaluated by sensory evaluation method, using a 9-point Hedonic Scale.
11. Method of experimental experiment on pasteurization of Solanum hainanense Hance drinks
The Solanum hainanense Hance drink recipes being selected in the above experiment were pasteurized to increase the shelf life of products. Pasteurization temperature was measured at 950C, 1000C, 1050C, 1100C and pasteurization time took place at 10 minutes, 20 minutes and 30 minutes. The microbiological requirements after pasteurizing were evaluated to select appropriate pasteurization conditions for each product.
13. Statistical processing methods
Data was synthesized and processed by Microsoft Excel. Experimental results were analyzed by using One Way ANOVA and Minitab 16.20 software.
Results and Discussion
1. Result of determination of some main components in Solanum hainanense Hance Table 1. The content (%) of some main components in Solanum hainanense Hance
Component (%) Leaf Trunk Roof
Water 78.996 62.745 49.026
Ash 0.431 0.464 0.438
Reducing sugar 1.821 1.352 1.546
Starch 2.175 9.300 7.684
Cellulose 3.953 16.133 32.757
Protein 2.529 1.863 1.775
2. Result of surveying the effect of drying methods on the extraction process
Figure 1 Graph shows the absorption spectrum of Solasodine in the visible light region
Each compound has a spectral absorption spectrum within the different wavelengths.
Solasodine compounds mainly contained in the absorption spectrophotometer in the range of 190-400 nm. Looking at the chart we can see, the Solasodine compound absorbs the highest visible light at wavelength 206nm.
Figure 2 Result of surveying the effects of two methods of drying (a, b: different letters represent statistically significant difference with p ≤5%).
The optical density of extracted solution at the absorbance of 206nm was used to evaluate the existence of solasodine. The higher the optical density value, the higher the solasodine content in the extract. The results showed that the sample using the vacuum drying method had higher optical densities, compared to the convection drying method.
0.854a
0.588b
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Optical desity (206nm)
Drying methods
Convection drying Vacuum
drying
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3. Result of surveying the drying temperature affecting the extraction process
Figure 3 Result of surveying the drying temperature affecting the extraction process (a, b: different letters represent statistically significant difference with p ≤ 5%)
The figure 3 shows that the extraction of the sample at 500C recorded the highest optical density. This is explained by the fact that the solasodine compound could be metabolized or lost during drying process when the temperature rose.
4. Result of surveying the drying time affecting the extraction process
Figure 4 Line graph illustrates the dependence of water content in material during drying time (a, b, c: different letters represent statistically significant difference with p ≤ 5%)
1,007a
0,836b
0,722c
0 0.2 0.4 0.6 0.8 1 1.2
50 55 60
Optical density (206nm)
Temperature (oC)
16.925a
9.340b
5.058c 4.637c
0 2 4 6 8 10 12 14 16 18
Water content (%)
Drying time (hours)
UV
5 6 7 8
Water content
1.007
a0.836
b0.722
cFigure 5 Result of surveying the drying time affects the extraction process (a, b, c: different letters represent statistically significant difference with p ≤ 5%)
The chart in Figure 4 shows the moisture content of the material during drying process for a period of 8 hours compared to that of the drying time in the 7-hour period decreased unnegligibly, basing on the results of the OD measurements from the extraction of the samples at the time of drying process. The sample dried for 7 hours recorded the highest optical density. Therefore, we chose a drying time of 7 hours.
5. Result of surveying the effect of solvent on solasodine extraction from Solanum hainanense Hance
Figure 6 Result of surveying the effect of solvent on solasodine extraction from Solanum hainanense Hance (a, b, c: different letters represent statistically significant difference with p ≤ 5%)
From the results obtained, the highest solasodine content was found for solvent ethanol (0.730).
The difference in solvent extraction efficiency could be explained as: solvent extraction efficiency depends on the ability of the solvent to diffuse deep inside the material layer. Extracting solvents affect the solubility of other compounds. Besides, solasodine is a very weak polar compound, it is soluble less in water. In the same condition, ethanol 95% obtained the best solasodine extract results. On the other hand, solasodine is mainly used in medicine to treat and ethanol is a non-toxic solvent, which is safe for human health, easy to find and cheap. Based on the results, we decided to choose ethanol as
0.772c
0.716c
0.846a
0.814ab
0.65 0.7 0.75 0.8 0.85 0.9
Optical density (206nm)
Drying time (hours)
0.730a
0.486b
0.232c
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Ethanol 95% Acetone Nước
Optical density (206nm)
Solvent
5 6 7 8
Ethanol 95% Acetone
Water
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solvent to extract solasodine.
6. Result of surveying the effects of material/solvent ratio on extraction process
Figure 7 The graph shows the dependence of the optical density on the material/solvent ratio of 1/4 (S1), 1/5 (S2), 1/6 (S3), 1/7 (S4), 1 1/8 (S8), corresponding to the amount of solvent (20, 25, 30, 35, 40, 45, 50 and 75ml)(a, b, c, d, e: different letters represent statistically significant differences with p ≤ 5%)
The result in Figure 7 shows that: the ratio of material / solvent significantly influences solasodine extraction. This is shown in detail by varying the OD206nm value at different scales.
When increasing the amount of solvent, the value of OD206nm also grew. However, this value only increased to a certain limit. The optical density rose very slightly at ratio from 1/15 to 1/10, so we decided to choose 1/10 to conduct the next experiment.
7. Result of surveying the effects of temperature on the extraction process
Figure 8 Result of surveying the effects of temperature on the extraction process (a, b, c: different letters represent statistically significant differences with p ≤ 5%)
0.439e 0.491de 0.540cde 0.542cd
0.615bc
0.690b
0.802a 0.834a
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Optical density (206nm)
Material/Solvent
0.621c
0.686c 0.705bc
0.925a
0.799b
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Optical density (206nm)
Temperature (0C)
S1 S2 S3 S4 S5 S6 S7 S8
40 45 50 55 60
60
When the temperature was low, the extraction was not effective, high temperature enhanced other reactions that were not necessary for the technological process. From Figure 8, we chose a temperature of 550C with the highest optical density value as the solasodine extraction temperature.
8. Result of the effect of extracting time on the extraction efficiency
Figure 9 Result of the effect of extracting time on the extraction efficiency (a, b: different letters represent statistically significant difference with p ≤5%)
Results showed that the sample extracted during 4 hours got the highest optical density.
This can be explained by the fact that raw materials in the form of powder were simultaneously treating in a solvent, along with the effects of temperature and the appropriate time, thereby disrupting the binding of the substances present in the cellulose. Solasodine was released and dissolved into solvent easily. As the extraction time increases within limits, the substances need time to dissolve and diffuse out of the material, so that the amount of extractable substance would be high. However, this relationship was linear within a certain limit. When the extraction time was prolonged under relatively high temperature conditions, the solasodine compound would be degraded. After a while, the substances contained in the material could no longer be dissolved in the solvent, and the dissolution of the solvents gradually decreased and the evaporation process increased. Thus, extending the extraction time would affect the efficiency of the technological process.
9. Result of the qualitation of solasodine by HPLC
Figure 10 Chromatogram HPLC of standard specimen (0.5 mg/mL) [6]
0.869b
1.002a 0.977a
0.8 0.85 0.9 0.95 1 1.05
Optical density (206nm)
Extracting time (hours)
3 4 5
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Figure 11 Chromatogram HPLC of test specimen
With the same conditions of conducting the HPLC analysis in the test specimen and standard specimen, we obtained the results shown in Figure 11, the retention time of the sample peak was equivalent to the retention time of the standard reference peak. Therefore, we conclude that the extract contains solasodine.
10. Result of experimental method for investigating the formula of beverage from Solanum hainanense Hance
Figure 12 depicts the sensory evaluation of the state, color, odor, and taste of the concentrated solasodine solution in 100 ml of water. The results show that the level of preference for the product tends to increase from 0.25 ml of the concentrated solasodine solution to the highest of 1 ml of solasodine. After that, when the concentration of solasodine increased from 1.25ml to 1.5ml, the level of interest tended to decrease. However, the sensory score at later major milestones was still higher than the levels of 0.25 ml - 0.75 ml.
At solasodine volume ratios of from 0.25 to 0.75 ml, the product state achieved the clarity. However, the color was too light, the green of Solanum hainanense Hance was not visible, the aroma was not clear and the taste was pale. At a rate of 1ml, the sensory score was highest because the product obtained the standard of clarity, dark green, pleasant aroma of Solanum hainanense Hance, an acceptable taste as well as sweet and easy to drink. Increasing product solasodine content (1.25ml and 1.5ml) reduced the likelihood of the product to drop relative to the 1ml of concentrated solasodine, as the product had a strong flavor, a heavy taste, and a slightly bitter pylorus.
From the above results, the sugar-free formula of Solanum hainanense Hance drink was selected with ratio at 1: 100 (concentrated solasodine: water) and this formula was used for further experiments.
Figure 12 The graph shows the difference in sensory value in formulations of concentrated solasodine in 100 ml of sugar-free Solanum hainanense Hance drinks
11. Result of surveying the sugar and citric acid content in the formulation of sugary Solanum hainanense Hance drinks
Figure 13 shows the overall sensory point of the Solanum hainanense Hance drinks with sucrose concentrations between 1% and 6% relative to citric acid concentrations of 0.01%-0.04%.
Result of statistical analysis showed that when we remained the citric acid content of 0.01% and 0.02%, the level of product preference did not differ from the level of significance α <0.05 for formula with sugar content ranging from 1% to 6%. Meanwhile, there was a difference (α <0.05) in preference to fixed formulas of citric acid content between 0.03% and 0.04% relative to the sugar concentrations ranging from 1% to 6%. Specifically, when we fixed the citric acid content at 0.03%, the formula containing a sugar concentration from 3% to 6% recorded the higher sensory evaluation score, compared to that of the low sugar formula of 1% -2%. Similarly, when we fixed citric acid content at 0.04%, the formula with higher sugar concentration (3%-6%) had a higher sensory evaluation score than the low sugar formula (1%-2%).
The graph (Fig. 13) illustrates that the formula containing 4% sugar with the citric acid content of 0.03% and 0.04% gave a higher sensory value compared to the remaining formulas. In addition, the results of statistical analysis showed that there was no significant difference in the significance level of α <0.05 with respect to both formulas. Therefore, the formula for Solanum procumbens drinks containing 4% sugar and 0.03% citric acid was selected.
0 1 2 3 4 5 6 7 8
0.25 0.50 0.75 1.00 1.25 1.50
Sensory evaluation score
Concentrated solasodine content in 100ml free-sugar Solanum procumbensdrinks (ml)
Texture Color Flavor Taste
Overall evaluation
Concentrated solasodine content in 100ml free-sugar Solanum hainanense Hance drinks
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Figure 13 Graph shows the difference in sensory values of formulas adding sugar and citric acid in Solanum hainanense Hance drinks
12. Result of surveying the formula of Solanum hainanense Hance drinks adding isomalt sugar
The formula of Solanum hainanense Hance drinks containing dietary sugar were established by adding about 5%-30% isomalt and 0.03% citric acid into sugar-free products.
Results of sensory evaluation are shown in Figure 14.
When the isomalt content increased from 5% to 20%, the preferred level was likely to increase in texture, color, flavor and taste indexes. In it, the sensory score of drink that added 20% isomalt registered the highest figure. However, when the sugar content increases to 25% and 30%, the level of preference tended to decrease after reaching a peak at 20% and the sensory scores remained above 5 points.
According to the description, the product that added 20% of isomalt sugar content met the demand the standard of clarity with a slightly yellow color, the smell of Solanum procumbens combined with the smell of sugar, thereby creating good taste and sour taste in harmony. As a result, the Solanum hainanense Hance drink containing 20% of isomalt sugar and 0.03% was selected.
Figure 14 Graph shows the difference in sensory values of formulas adding substitute sugar in Solanum hainanense Hance drinks
0 1 2 3 4 5 6 7 8
1 2 3 4 5 6
Sensory evaluation score
Sugar content (%)
Citric acid 0.01%
Citric acid 0.02%
Citric acid 0.03%
Citric acid 0.04%
0 1 2 3 4 5 6 7 8
5 10 15 20 25 30
Sensory evaluation score
Isomalt content(%)
Texture Color Flavor Taste
Overall evaluation
Sugar content (%)
13. Physical and chemical properties (soluble dry matter and pH value) of Solanum hainanense Hance drinks before and after pasteurization
The values of the dry matter content and pH of the three products of Solanum hainanense Hancedrinks (sugar-free, sugary, substitute sugar) before and after pasteurization are shown in Table 2. The results show that the selected pasteurization regime did not alter the dry matter content and the pH value of the products. In addition, the products were acidic. Particularly, the sugary produtcs and products containing substitute sugar witnessed low pH values at roughtly 3.1 and 3.9, respectively.
Table 2 Physical and chemical properties (soluble dry matter and pH value) of Solanum hainanense Hance drinks before and after pasteurization
The physical and chemical properties
Solanum hainanense Hance drinks
Sugar-free Sugar Sugar substitute Soluble dry matter
content (%)
Before pasteurization
0.5 5.0 12.0
After pasteurization
0.5 5.0 12.5
pH Before
pasteurization
6.1 3.0 4.0
After pasteurization
6.1 3.1 3.9
14. Results of sensory evaluation of Solanum hainanense Hance drinks
The selected formulations of Solanum hainanense Hance drinks in the above experiments were pasteurized and evaluated to determine the interest of customers (Table 3). The results showed that the addition of sweeteners (sugars or substitute sugar) enhanced the preference of consumers, compared to sugar-free products in terms of taste. However, there was no difference (α <0.05) in color, odor and state between sweet products and sugar-free ones. This indicates that sweetened Solanum hainanense Hance drinks were more popular than sugar-free ones.
Table 3 Results of sensory evaluation of Solanum hainanense Hance drinks
Solanum hainanense Hance drinks Color Flavor Taste Texture
Sugar free 5.30ab 5.75a 3.25a 5.20a
Sugar 4.75a 5.75a 580b 5.50a
Sugar substitute 5.20ab 5.55a 5.20b 5.35a
Conclusion
Solasodine is a valuable bioactive compound in Solanum hainanenense Hance. The study found the optimum method for crude solasodine extraction including drying mode (drying method, drying time, drying temperature) and the extraction method (type of solvent, ratio of material/solvent, extraction temperature, and extraction time). Inheriting the results obtained from the extraction process, the formulas for beverages from Solanum hainanenense Hance extraction were established and reached a certain preference for the sensory tester.
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From the results obtained in this work we concluded that the Solasodine compound absorbs the highest visible light at wavelength 206nm and the sample using the vacuum drying method had higher optical densities, compared to the convection drying method. The drying process was effective at 50 C in 7 hours. The extraction used ethanol 95% with the ratio of 1/10 (material/souvent) at 55 C in 4 hours. The sugar-free Solanum hainanense Hance drinks was mixed with ratio at 1: 100 (concentrated solasodine: water). The formula for Solanum hainanense Hance drinks containing 4% sugar and 0.03% citric acid recorded the highest points. Besides, the Solanum hainanense Hance drinks containing 20% of isomalt sugar and 0.03% was prefered. This indicates that sweetened Solanum hainanense Hance drinks were more popular than sugar-free ones.
References
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