**3. Result**

In the α-amylase inhibitory activity test, the amount of soluble starch decreased by the enzyme reaction was measured by the iodine reaction method. The inhibitory activity of PM extract against pancreatic α-amylase was determined using different concentrations (1.875, 3.75, 7.5, 15, and 30 mg/mL). As shown in Table 1, the PM extract inhibited the enzyme activities in a dose-dependent way.


**Table 1.** α-Amylase inhibitory activity of PM (*Phaseolus multiflorus* var. albus Bailey) extract.

> Data are presented as medians ± standard deviations (n = 3).

Pancreatic lipase is the most important enzyme for the digestion of dietary triacylglycerols. Pancreatic lipase is a key enzyme that hydrolyzes 50% to 70% of total dietary fat in the digestive system, converting TGs to monoglycerides and free fatty acids [18]. Inhibiting pancreatic lipase is an important strategy for treating obesity and other metabolic disorders [19]. In this study, PF extract inhibited pancreatic lipase activity in a concentration-dependent manner, with an Inhibitory concentration (IC50) value of 1.68 mg/mL (Table 2). The lipase inhibitory activity of PF extract may be able to suppress dietary fat absorption in vivo as well.

**Table 2.** Pancreatic lipase (PL) inhibitory activity of PF (*Pleurotus eryngii* var. ferulae) extract.


Data are presented as medians ± standard deviations (n = 3).

As a result of in vitro tests, the effect of inhibiting alpha amylase of PM extra and the effect of inhibiting pancreatic lipase of PF extra were the highest at 15 and 5 mg/mL, respectively. Based on the in vitro test results, a DKB-117 extract mixed with a PM:PF = 3:1 (w/w) ratio was prepared to perform in vivo tests by concentration (100, 200, and 300 mg/kg).

The results of the eight weeks of body weight measurement showed that the HFD-treated group had significantly higher values than the ND-treated group, while the DKB-117-300-, orlistat-, and *Garcinia cambogia*–treated groups had significantly decreased weight as compared with the HFD-treated group (*p* < 0.001). The administration of DKB-117-300 was considered to have affected the reduction of body weight in obese models induced by HFD (Figure 1).

The concentrations of TCHO, TGs, and low-density lipoprotein (LDL) in the serum were also remarkably higher in the HFD group compared to those in the normal diet (ND) group, which is also a typical symptom of obesity [20].

As shown in Table 3, the HFD induced hyperlipidemia, with increases of plasma triacylglycerol and cholesterol levels. The serum levels of total cholesterol (TCHO), TGs, high-density lipoprotein (HDL), and LDL sharply increased in the HFD group compared to those in the ND group. LDL level was significantly lower (*p* < 0.05) than in the HDF group and reduced by 19.7% after eight weeks of DKB-117-300 administration. No significant alteration was observed in the serum levels of TCHO and TGs. However, these conditions were improved after eight weeks of DKB-117-300 administration (Table 3). These data clearly indicate that DKB-117 intake can effectively mitigate hyperlipidemia

induced by HFD by reducing the lipid content in the blood. Furthermore, it was shown that the effects of weight gain reduction by DKB-117-300 treatment had an overall effect on the blood lipid metabolism index.

**Figure 1.** Effects of DKB-117 extracts on body weight in high-fat diet (HFD)–induced obese mice. The values are presented as means ± standard error of the means (SEMs). \*\*\* A significant difference at the *p* < 0.001 level was observed versus the normal diet group (ND). ### A significant difference at the *p* < 0.001 level versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.

**Table 3.** Effect of DKB-117 extracts on serum lipid profiles in HFD-induced obese mice after fasting for 15 h at the end of the study.


The values are presented as means ± SEMs. \*\*\* A significant difference at the *p* < 0.001 level was observed versus the ND. # A significant difference at the *p* < 0.05 level was observed versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg, TG: triglyceride; HDL, high-density lipoprotein, LDL: low-density lipoprotein, TCHO: total cholesterol.

Sung et al. reported that extracts exhibiting lipase inhibitory activity effectively lower cholesterol and TG levels that were elevated due to HFD intake. In this experiment, the lipase inhibitor (orlistat) effectively lowered TCHO, TGs, and LDL, which are increased by HFD intake [19]. Orlistat is known to inhibit the hydrolysis of dietary fat to free fatty acids so that the fat is not absorbed in the intestine but rather is excreted directly in the feces, thereby reducing a person's weight and improving their blood lipid levels and glucose metabolism [21]. The DKB-117-300-treated group appeared to present improvements in the lipid metabolism index effectively through lipase inhibitory activity.

Micro-CT scans showed that the total fat, abdominal fat, and subcutaneous fat were all increased in the HFD-induced obese mice compared with those in the ND mice. However, these fats were significantly decreased when DKB-117-100 administration was concomitant. Especially, DKB-117-100 showed an equal or superior effect to that of orlistat and *Garcinia cambogia* used as positive controls. In the case of orlistat, only the visceral fat amount was significantly higher than that in the HFD-treated group (Figure 2).

The experiment was carried out during eight weeks. We measured the weight of inguinal and abdominal fat. After finishing the eight-week test, the weights of inguinal fat and abdominal fat, respectively, were measured after the autopsy. The orlistat group showed a statistically significant decrease in the inguinal fat mass compared to the HFD group, while the DKB-117-100 group showed a statistically significant decrease in the absolute amount of abdominal fat and inguinal fat compared to the HFD group (Figure 3).

(**A**)

**Figure 2.** *Cont*.

**Figure 2.** Effects of DKB-117 extracts on fat volume in HFD-induced obese mice. C57BL/6J mice consumed a HFD or ND for eight weeks. (**A**) Transverse microcomputed tomography (micro-CT) images. (**B**) Total fat volume, (**C**) abdominal fat volume, and (**D**) subcutaneous fat volume were measured using micro-CT. The values are presented as means ± SEMs. \*\*\*/\*\*/\* A significant difference at the *p* < 0.001/*p* < 0.01/*p* < 0.05 level was observed versus the ND. # A significant difference at the *p* < 0.05 level was observed versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.

(**D**)

**Figure 3.** Effect of DKB-117 extracts on (**A**) abdominal and (**B**) inguinal fat weight in HFD-induced obese mice. The values are presented as means ± SEMs. \*\*\*/\*\*/\* A significant difference at the *p* < 0.001/*p* < 0.01/*p* < 0.05 level versus the ND. ##/# A significant difference at the *p* < 0.01/*p* < 0.05 level versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.

As a result of the measurement of fat content in the liver using Oil-Red-O staining, the HFD-treated group revealed a significant increase in fat area in the liver compared to the ND-treated group (*p* < 0.001). In the DKB-117-treated group (100, 200, or 300 mg/kg), the dose-dependent area of fat staining decreased, and the DKB-117-100-treated group showed a significant decrease compared to the HFD-treated group (*p* < 0.05). In addition, the DKB-117-100-treated group showed statistically equivalent efficacy results compared to the *Garcinia cambogia*–treated group (Figure 4).

**Figure 4.** Effect of DKB-117 extracts on lipid accumulation in HFD-induced obese mice. (**A**) The Oil-Red-O staining in liver sections (×400, magnification) and (**B**) quantification of the positively stained area. The values are presented as means ± SEMs. \*\*\*/\*\*/\* A significant difference at the *p* < 0.001/*p*<0.01/*p*<0.05 level compared to the ND. ###/# A significant difference at the *p* < 0.001/*p* < 0.05 level versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.

The liver is an important organ responsible for lipid metabolism, along with fat tissue [22]. Free fatty acids are the most basic elements of the energy metabolism in the body and are transferred to other organs in the form of TGs [23]. An imbalance in lipid metabolism causes an intracellular accumulation of TGs. We confirmed that liver fat was significantly accumulated by eating HFDs.

As shown in Table 3 and Figure 5, the HFD induced hyperlipidemia, with increases in plasma triacylglycerol and cholesterol levels and induced fatty liver along with an accumulation of triacylglycerol in the liver.

**Figure 5.** Effect of DKB-117 extracts on liver TG levels in HFD-induced obese mice. The values are presented as means ± SEMs. \*\*\* A significant difference at the *p* < 0.001 level versus the ND. ##/# A significant difference at the *p* < 0.01/*p* < 0.05 level versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.

As a result of the analysis of TG content in liver, the HFD-treated group showed a significant increase (*p* < 0.001) as compared with the ND-treated group, while the DKB-117-100-, orlistat- and *Garcinia cambogia*–treated groups showed a significant decrease as compared with the HFD-treated group (*p* < 0.05 or *p* < 0.001). Similar to the results of weight loss, the results here showed that DKB-117-300 intake reduced liver lipid accumulation through a mechanism of digestive enzyme inhibition (Figure 5).

The amount of carbohydrates in the feces was not different between the HFD and ND groups. The DKB-117-100 group showed a significant increase (*p* < 0.01) in carbohydrate content as compared with that in the HFD group, while the orlistat group showed a significant increase in TGs, TCHO, and carbohydrates at the eighth week (Figure 6).

**Figure 6.** Effects of DKB-117 extracts on feces carbohydrate levels. The values are presented as means ± SEMs. \*\*\*/\* A significant difference at the *p* < 0.001/*p* < 0.05 level versus the ND. ###/## A significant difference at the *p* < 0.001/*p* < 0.01 level versus the HFD. ND, normal diet control group; HFD, high-fat diet control group; DKB-117-300, DKB-117 300 mg/kg; DKB-117-200, DKB-117 200 mg/kg; DKB-117-100, DKB-117 100 mg/kg; Orlistat, orlistat 40 mg/kg; Garcinia, *Garcinia cambogia* 200 mg/kg.
