4.2.1. Selecting the best solvents for extracting total flavonoid from the Polyscias fruticosa trunk.
The solubility and constancy of compounds are influenced by solvents with antioxidant activity like flavonoids and polyphenols. Through extraction procedure, certain unwanted components may solube in the extract, altering the experimental results. As a result, choosing the appropriate solvent is critical to ensuring the greatest extraction of flavonoids.
To study the extraction ability of total flavonoids in Polyscias fruticosa trunk, distilled water and ethanol were used in this study. There are two kinds of solvents which are commonly used to seperate the constituents because they are inexpensive, easy to find, and easy to recover for reuse. Experiments were conducted with two formulations of solvents, respectively distilled water (H2O), ethanol (C2H5OH) 70% with a time of 20 hours and a solvent/material ratio of 10/1 (ml/g) at room temperature, then determine the flavonoid content, the results obtained are shown in the figure 8 below:
Figure 8. Effect of extraction solvent on the extraction of total flavonoids
32 Note: the letters a, b represent the significant difference at the 5% significance level.
From Figure 8 we see that the flavonoid content in each solvent is different. To be more specific, extraction with ethanol gives the highest flavonoid content 0.293±0.007 (mg/g), while distilled water provides the smallest amount of flavonoid content at 0.132± 0.003 (mg/g).
The results show that when statistically processed, the sample extraction with ethanol has a difference compared to the distilled water solvent at the 5%
level of significance. This is explained by the fact that total flavonoids can contain many non-polar components. It is hard for them to dissolve in polar solvents. Besides, in our research projects, ethanol was the better solvent because of its quick denaturation effect, destroys cell membranes, produces the best extraction conditions for the process of penetration and contacts with antioxidant active substances. Ethanol is rated as a highly safe, non-toxic solvent. Therefore, ethanol is the most appropriate solvent for the extraction of total flavonoids from the Polyscias fruticosa trunk.
4.2.2. The effect of concentration of extraction solvent on the total flavonoid content
Diffusion is a critical physical modify that happens during the extraction process. dissolved molecules will move from the center of materials to the surface region, and then from the surface to the solvent. Solvent molecules will be diffused from the outside to the inside of the material. Diffusion will speed up and fully remove the components to be extracted from the raw materials into the solvent. The main factor underlying diffusion is the concentration difference.
Ethanol concentrations have a significant effect on flavonoid solubility and total flavonoid content (Le Van Viet Man et al., 2011). To estimate the extraction capability of total flavonoid content, this experiment used ethanol solvent at concentrations of 70%, 80%, and 90%. The dried trunk material is immersed for 20 hours at room temperature in ethanol at 70%, 80% and 90%. The results are depicted in Figure 9 below:
33 Figure 9. The effect of extraction time on total flavonoid extraction
Note: the letters a, b, c represent the significant difference at the 5% significance level.
Figure 9 shows that the ethanol content increased, the flavonoid content also increased with the addition of solvent. Total flavonoids content obtained when using ethanol solvent when extracted at concentrations of 70%, 80% and 90% (v/v) were 0.296±0.002, 0.331±0.008, 0.419±0.021 mg/g respectively.
The highest flavonoid concentration was obtained at 90% ethanol concentration, much higher than 80% concentration. Meanwhile, when the concentration was 70%, the flavonoid content began to show a slight decrease.
As a result, ethanol with concentration at 90% is suitable for flavonoid extraction with the highest yield.
4.2.3. The effects of extraction time on the total flavonoid content
The extraction time is critical in the extraction of flavonoids. If the time is insufficient, the solute will not be completely dissolved in the solvent. When a certain time limit is reached, the amount of product obtained increases insignificantly, which may have an adverse effect on product quality due to the possibility of reverse osmosis of the solute into the raw materials or the decomposition of a compound.
34 In this experiment, the stem powder was soaked in ethanol at 90% for different periods of 16, 18, 20, 22 and 24 hours at room temperature. The results of the survey are illustrated in Figure 10:
Figure 10. The effect of extraction time on the total flavonoid content Note: the letters a, b, c represent the significant difference at the 5% significance level.
As shown by the results in Figure 10, the flavonoid content was highest when the soaking time was 20 hours and lowest when it was 24 hours. When processing extraction statistics in 20 hours, there is a gradual difference when compared to time points of 16 hours, 18 hours, 22 hours, and 24 hours with a significance level of 5%. Total flavonoids content obtained when soaking during 20 hours was 0.424±0.005, while when soaking during 16, 18, 22 and 24 hours, the total flavonoid content were 0.383±0.003, 0.388±0.003, 0.374±0.015 and 0.318±0.003 mg/g respectively. Thus, the results show that it is possible to extract with 90%
ethanol in 20 hours to improve total flavonoids extraction efficiency.
35 4.2.4. The effects of ratio between solvent and material on the total flavonoid content
The flavonoid content obtained is affected by the ratio between solvent and material. When the difference between the amount of solvent and the quanitive of material is too small, the connection between them is limited, and the concentration distinction between the solvent and the material is not enormous, limiting the diffusion ability of material. It is impossible to extract all of the compounds contained in the raw materials due to the dispersion of the compounds extraction to the solvent. However, once the extraction solution has reached equilibrium, the yield of extracted compounds will no longer increase (Herode et al., 2003). To obtain the highest flavonoid content, we conducted survey experiments with the following solvent/material ratios: 5/1, 10/1, 30/1, 50/1, 70/1, and 100/1 (ml/g). Figure 11 represents the results of an experiment in which 90% ethanol was soaked for 20 hours at room temperature.
Figure 11. The effect of solvent/material ratio on the total flavonoid content Note: the letters a, b, c, d represent the significant difference at the 5%
significance level.
Figure 11 shows that the highest flavonoid content was obtained when the solvent/material ratio was 10/1 (ml/g). At the ratio of 10/1 (ml/g), the concentration of total flavonoid content reached at 0.432 ± 0.004 (mg/g). In
36 constrast, the flavonoid content decreased significantly when extracted at different ratios including 1/5, 1/30, 1/50, 1/70, and 1/100. In particular, when extracted with the different ratios, the flavonoid content was 0.348 ± 0.005, 0.320
± 0.003, 0.320 ± 0.005, 0.313 ± 0.004 and 0.297±0.005 (mg/g). At the significance level of 5%, extraction with the ratio between solvent and material of 10/1 (ml/g) differed from the other solvent/material ratios.
As a result, we chose a solvent/material ratio of 10/1 (ml/g) to achieve both extraction efficiency and solvent savings.
4.2.5. The influence of raw material size on extraction capability of total flavonoid content
The material's size and shape are a constraint. The size of the raw material influences the contact area between the material and the solvent, which in turn influences the extraction procedure. In general, the higher the extraction capacity, the smaller the material size, due to the increased contact area between the solvent and the material. As a result, the solvent easily penetrates the material and diffuses the solutes out. In this experiment, we sieved through three sieve hole diameters:
1mm or less, 1 to 3mm, and 3 to 5mm, before immersing the sample in 90% ethanol for 20 hours at a 10/1 (ml/g) ratio to determine the difference when extracting at various sizes. The results are illustrated in Figure 12 below:
Figure 12. The influence of Material size on extraction capability of total flavonoid content
Note: the letters a, b represent the significant difference at the 5% significance level.
37 Figure 12 demonstrates that the flavonoid content varies with size. It can be seen that if the size is larger, the extraction capability will be low. To be more specific, when extracted with each sizes, the flavonoid content were 0.450±0.012, 0.351±0.02, 0.385±0.012 (mg/g) respectively. The highest flavonoid content was obtained at 0.450±0.012 (mg/g) in fine powder with a size less than or equal to 1mm. When statistics are processed, extracting with sizes less than or equal to 1 mm differ from those with sizes ranging from 1 to 3 mm or 3 to 5 mm at the significance level of 5%.
As a result, in order to maximize extraction efficiency, size of 1 mm or less is the best choice for the next experiment.
4.2.6. The effect of magnetic stirring on the extraction capacity of total flavonoid content
The mixing of the raw materials has a significant impact on extraction efficiency because the diffusion of essential oils in the raw materials into the solvent is facilitated by frequent mixing of the raw materials and the solvent. As a result, we investigated the effect of magnetic stirring on the ability to extract flavonoids at a rate of 10/1 ml/g while immersing at room temperature during 20 hours (stirring 5 hours each time) to see if the total flavonoid content was greater or lower. The following image 13 shows the test results:
Figure 13. The influence of magnetic stirrer on the total flavonoid content
38 Note: the letters a, b represent the significant difference at the 5% significance level.
Figure 13 shows that the total flavonoid content under the effect of magnetic stirrer is 0.557 ± 0.005 mg/g and of no magnetic stirrer is 0.452±0.018 mg/g. After analytical processing, the results between the effects of magnetic stirring and no magnetic stirring have a difference at the 5% significance level.
As a result, in order to maximize extraction efficiency, we chose to use magnetic stirrer to contribute to the extraction process of total flavonoid.