## 4 Discussion  
In this study, we focused on the optimal hydrothermal extraction conditions for _A. tegmentosum_ to maintain maximal antioxidant activities in vitro. There are several reports on using hydrothermal extraction methods with _A. tegmentosum_ for extracting phenolic compounds [22; 23]; however, the optimal extraction methods are not clearly understood. Therefore, we determined the optimal hydrothermal extraction conditions for these individual extraction factors for _A. tegmentosum_. Unlike the experiments that use one factor at a time, the extraction time and the solvent-to-solid ratio were set outside the setting range for our CCD experiment. In this case, the extraction temperature might be lower than the optimum range due to physical constraints; the conditions should be adjusted accordingly by increasing the extraction temperature or the solvent-to-solid ratio. Increasing the extraction temperature provides physical energy to isolate functional compounds from the raw materials. Moreover, the elevation of the solvent-to-solid ratio also increases the extraction yield of functional compounds by increasing their surface area. The results of our CCD indicate a rough optimal hydrothermal extraction area for _A. tegmentosum_. If functional materials are altered by metabolism, decomposition, and/or oxidation after extraction, the CCD may not fully explain the optimal extraction conditions. [PERSON] et al. [24] also used a CCD to determine optimal coal ash extraction conditions by altering the extraction temperature, the extraction time, and the solvent-to-solid ratio. In this study, the CCD solvent was the main factor affecting the extraction outcome; the extraction time and temperature did not have a significant effect on the outcome. Therefore, a CCD is a good indicator not only for determining optimal extraction conditions but also for understanding the extraction conditions that significantly affect the outcome. As shown in previous studies, many functional compounds are present in the _Acer_ genus, such as phenolic compounds, including flavonoids, salidroside, gallic acid, and catechin [25; 26; 27]. LC-Q-TOF-MS analysis revealed the presence of three catechin families (epicatechin, catechin-(4\(\alpha\)-\(>\)8)-epicatechin-3-O-gallate, and (-)-epicatechin-3-O-gallate). Catechins are plant-based flavonoid materials that contain various functional ingredients in green tea and cocoa [24]. Catechins have been reported to have insulin-sensitizing and blood-lipid-lowering effects and protect against cardiovascular disease [3]. Moreover, epicatechin series such as epicatechin, epigallocatechin, and epigallocatechin gallate [28; 29; 30] are sometimes  
Figure 7: Cell viability and reactive oxygen species (ROS) production in HepG2 cells were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and the 2,7-dichlorourescin diacetate (DCF-DA) method. The results are expressed as a percentage of the control (as 100%), and each bar graph represents the mean \(\pm\) standard deviation (\(n=3\)). HepG2 cells were pretreated in the absence (sterile water for control) and presence of _A. tegmentosum_ extract (0-40 \(\mu\)g/mL) for 1 h, and 1 mM H\({}_{2}\)O\({}_{2}\) was added for 24 h. (**A**) Hepatic cellular viability was measured by MTT assay, and (**B**) ROS production was assessed in cultures by the DCF-DA method. Data are presented as the mean \(\pm\) standard deviation (\(n=3\)). Labeled averages without a common letter differ. \(p<0.05\). lost by heating; therefore, it is necessary to find the optimal extraction conditions for edible plants to maximize their functional properties [31]. Salidroside is another major functional compound in _A. tegmentosum_. Salidroside has a glucoside of tyrosol, which is generally soluble in nonpolar solvents such as methanol and ethanol. Salidroside has anti-inflammatory and antioxidative effects; therefore, hydrothermal extraction may extract other antioxidative compounds and compensate for the role of salidroside [6,26].