## 3 Results and Discussion
### Solvent Fractionation for Antimutagenic Active Compound Group  
The fractionation was carried out with the 70% ethanol FFL extract to identify major active compound groups. The scheme and yield of the separation and purification processes are shown in Figure 1. The dried FFL powder (500 g) was extracted in 70% ethanol, and the extraction yield was 34 g. Thereafter, solvents with five different polarities were used to generate five fractions. The 70% ethanol extract was fractionated into diethyl ether (14.2 g), chloroform (1.25 g), dichloromethane (1.3 g), butanol (6.68 g), and water (7.9 g) fractions, respectively. Then, each fraction was employed for the antimutagenicity test (Figure 5). As occurred with the result of the previous section (Figure 4), no antimutagenicity was observed without the S9 mix in both strains. In the TA98 strain, all extracts exhibited a dose-response inhibition in the presence of the S9 mix, except for dichloromethane (Figure 5a). The IC\({}_{50}\) values for each FFL extract were 505.3, 3223, 525.3, and 2811 ug/plate for diethyl ether, chloroform, butanol, and water, respectively. They were ranked in the following order: diethyl ether \(\geq\) butanol \(>\) water \(\geq\) chloroform. In the TA100 strain, diethyl ether, chloroform, and butanol exhibited a dose-response inhibition with the S9 mix (Figure 5b). The IC\({}_{50}\) values were 756.4 and 906.3 ug/plate for diethyl ether and butanol, respectively. They were ranked in the following order: diethyl ether \(\geq\) butanol \(>\) chloroform. Robust antimutagenic effects were observed in the diethyl ether and butanol fractions, which implies that they possess bioactive compounds. In general, solvent fractionation is applied in natural product research primarily to separate and characterize specific compounds or groups of compounds from complex mixtures [42]. This method is particularly useful for identifying active ingredients from natural sources, especially when natural products with diethyl ether, such as essential oils, terpen, alkaloids, steroids, and lipid-soluble vitamins, are primarily extracted [43]. Some compounds of these groups revealed antimutagenic effects [44; 45; 46]. The essential oils extracted from _Ferula orientalis_ L. exhibited antimutagenic effects in _S. typhimurium_[44]. The essential oils and monoterpenes extracted from _Salvia officinalis_ L. also showed antimutagenic effects [45]. The isolated one triterpen and four steroids from the thorns of _Gleditsia sinensis_ L. caused an inhibitory effect against mutagens using the _E. coli_ PQ37 strain [46]. Lipid-soluble vitamins such as vitamins A and E were also reported to possess antimutagenic effects [47; 48]. In the case of butanol fraction, butanol solvent is typically used to extract compounds with mid-to-high polarity [42]. The extract of butanol includes flavonoids, glycosides, and alkaloids [42]. It is particularly useful for separating compounds that are more polar than those soluble in water. Flavonoids were also reported to induce potent antimutagenic effects. The flavonoid structure contains the free 5-,7-hydroxyl group considered to be a major active  
Figure 5: Antimutagenicity effects of two strains by the fractionized FFL extracts in the presence of the S9 mix; (**a**) TA98 strain; (**b**) TA100 strain. The inhibitory effect was tested using 2-aminoanthracene (2-AA; 0.5 ug/plate). The inhibition rate is shown as means \(\pm\) SD. compound [49; 50]. Two chalcone glycosides isolated from _Mentha longifolia_ L. exhibited antimutagenic effects in the TA1537 strain [51]. In addition, [PERSON] et al. reported antimutagenic effects in quercetin glycosides [52]. Alkaloids such as quinolizidine alkaloids also exhibited antimutagenic effects [53; 54; 55]. Like this, many substances extracted from natural products present antimutagenic effects by preventing the activation of mutagens in or out of body cells, and the mentioned compound groups are commonly detected in mushrooms, including FFL [7; 23].