Anti-Obesity Effect of DKB-117 through the Inhibition of Pancreatic Lipase and α-Amylase Activity

This study sought to evaluate the effects of Phaseolus multiflorus var. albus Bailey extract (PM extract) and Pleurotus eryngii var. ferulae extract (PF extract) on the inhibition of digestive enzymes and to confirm the anti-obesity effect of DKB-117 (a mixture of PM extract and PF extract) in digestive enzyme inhibition in a mouse model of obesity induced by a high-fat diet. In in vitro studies, PM extract and PF extract have increased dose-dependent inhibitory activity on α-amylase (Inhibitory concentration (IC50 value: 6.13 mg/mL)) and pancreatic lipase (IC50 value; 1.68 mg/mL), respectively. High-fat diet–induced obese mice were orally administered DKB-117 extracts at concentrations of 100, 200, and 300 mg/kg/day, while a positive control group was given orlistat (pancreatic lipase inhibitor) and Garcinia cambogia (inhibiting the enzymes needed to synthesize carbohydrates into fat) at concentrations of 40 and 200 mg/kg/day, respectively, for eight weeks. As a result, body weight, fat mass (total fat mass, abdominal fat, and subcutaneous fat) detected with microcomputed tomography, fat mass (abdominal fat and inguinal fat) after an autopsy, and liver triglyceride levels were decreased significantly in the DKB-117 (300 mg/kg/day) group compared to those in the HFD control group. Additionally, we obtained results indicating that the presence of carbohydrates was found more in the DKB-117-300 (300 mg/kg/day) group than in the HFD control group. These data clearly show that DKB-117 extracts are expected to have an anti-obesity effect through a complex mechanism that promotes carbohydrate release through the inhibition of carbohydrate-degrading enzymes while blocking lipid absorption through lipase inhibition.


Introduction
Obesity is a metabolic disorder characterized by an excess accumulation of fat in the body due to one's energy intake exceeding one's energy expenditure [1]. Obesity is a very common global health problem. It was reported by the World Health Organization in 2016 that more than 1.9 billion adults were overweight and, of these, more than 650 million were obese [2]. Obesity is a risk factor for metabolic syndrome and can lead to hypertension, type 2 diabetes (T2DM), dyslipidemia, cardiovascular disease (CVD), and stroke [3,4].

α-Amylase Inhibition Assay
The measurement of α-amylase inhibitory activity was carried out via the iodine reaction method as described by Wilson et al. with a slight modification [16]. Briefly, α-amylase (Sigma-Aldrich, St. Louis, MO, USA) derived from human saliva was dissolved in phosphate-buffered saline (PBS) at a concentration of 20 unit/mL. As a substrate for α-amylase, soluble starch was dissolved in PBS at a concentration of 1%. To measure the inhibitory activity against α-amylase, 290 µL of PBS, 10 µL of α-amylase solution (20 unit/mL), and 50 µL of the test substance were mixed and preincubation was performed at 37 • C for 10 min. After preincubation, 350 µL of 1% soluble starch as substrate was added and reacted at 37 • C for 30 min. To determine the amount of soluble starch remaining after the reaction, 300 µL of iodine solution (0.1% KI + 0.01% I 2 /0.05 N HCl) was added to the reaction solution and the absorbance was measured at 620 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Infinite 200 Pro; Tecan Austria GmBH, Grödig, Austria).

Pancreatic Lipase Inhibition Assay
Pancreatic lipase inhibitory activity was measured using the substrate p-nitrophenyl butyrate (PNPB) as described by Eom et al. with slight modification [17]. Briefly, an enzyme buffer was prepared by adding 30 µL of porcine pancreatic lipase (Sigma-Aldrich, St. Louis, MO, USA) in 10 mM of morpholinepropane sulfonic acid and 1 mM of ethylene diamine tetra acetic acid (pH: 6.8) to 850 mL of Tris buffer (100 mM of Tris-HCl and 5 mM of CaCl 2 ; pH: 7.0). Then, 100 µL of DKB-117 or orlistat was mixed with 880 mL of the enzyme buffer and incubated for 15 min at 37 • C. After incubation, we added 20 µL of the substrate solution (10 mM of PNPB in dimethyl formamide) and the enzymatic reactions were allowed to proceed for 30 min at 37 • C. Pancreatic lipase inhibitory activity was determined by measuring the hydrolysis of PNPB to p-nitrophenol at 405 nm with the use of an ELISA reader (Infinite 200 Pro; Tecan Austria GmBH, Grödig, Austria). The activities of the negative control were reviewed with and without the inhibitor. The inhibitory activity (%) was calculated according to the formula below: Lipase where A is the activity of the enzyme without the inhibitor, a is the negative control without the inhibitor, B is the activity of the enzyme with the inhibitor, and b is the negative control with the inhibitor.

Animal Experiments
The protocol for animal study was approved by the Department of Biofood Research, KNOTUS Life Science Co., Ltd. Animal Ethics Committee . Male C57BL/ JJmsSlc mice, five weeks old (18-20 g), were purchased from Central Lab Animal Inc. (Seoul, Republic of Korea). The animals were Nutrients 2020, 12, 3053 4 of 13 housed in polycarbonate cages (less than five mice/cage) under controlled temperatures (23 ± 3 • C), relative humidity (55% ± 5%), and lighting conditions (lights on from 08:00 to 20:00 h), with food and water made available ad libitum. Mice were fed either a regular diet (ND group; lab rodent chow; Cargill-Agri Purina, Seongnam-Si, Republic of Korea) or a 60% HFD (HFD group; Saeronbio, Uiwang-si, Republic of Korea). On the 19th day of HFD feeding, the body weight of the HFD group was 10% higher than that of the ND group. At this time, the HFD group was divided into six additional groups: an HFD control group, a DKB-117-100-treated group (100 mg/kg/day), a DKB-117-200-treated group (200 mg/kg/day), a DKB-117-300-treated group (300 mg/kg/day), an orlistat-treated group (40 mg/kg/day), and a Garcinia cambogia-treated group (200 mg/kg/day), respectively. For eight weeks, the mice were daily treated with test article and HFD, with the exception of the ND group.
After eight weeks of treatment, the mice were not fed for 15 h and their body weight was measured. Then, the mice were anesthetized with ether. Blood was drawn from the postcaval vein for serum biochemical analysis. Extracted liver, abdominal fat, and inguinal fat tissue were weighed.

Micro-CT Analysis
At the end of the treatment, micro-CT (viva CT80; SCANCO Medical, Switzerland) was conducted, and body fat mass was analyzed from the basal region of second lumbar to the distal end of the fifth lumbar.

ELISA Assay
After eight weeks of treatment, carbohydrate levels in collected fecal samples and TG levels in right-lobule liver tissue were measured. The analysis was carried out using a commercial ELISA kit (Biochemical, MI, USA, USA, Triglyceride Assay kit, Asanpharm, Korea, Triglyceride Colorimetric Assay Kit, Cayman, Chemical, USA).

Histopathological Analysis
Left-lobule liver tissue dissected from mice was fixed in 10% neutral-buffered formalin solution. The dehydrated liver tissue was then embedded in paraffin wax, and sections were cut from the paraffin-embedded tissues. These sections stained with Oil-Red-O. Histopathological changes were assessed using an optical microscope (BX53, Olympus, Tokyo, Japan) and image analyzer (Zen 2.3 blue edition; Carl Zeiss, Jena, Germany).

Statistical Analysis
The assumption of homogeneity was tested using Levene's test. If the overall analysis of variance was significant and the assumption of homogeneity of variance was met, Duncan's multiple range test was performed. If the assumption of homogeneity of variance was not met, Dunnett's T3 test was used as the post hoc test. In nonparametric multiple analysis, the Kruskal-Wallis H-test was adopted. If a statistically significant difference was observed between groups, the Mann-Whitney U-test was used to identify the groups. SPSS Statistics 18.0K (IBM Corp., Armonk, NY, USA) was used for all statistical analysis and the level of significance was set at p < 0.05.

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 Nutrients 2020, 12, 3053 5 of 13 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. 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 (IC 50 ) 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. 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 Nutrients 2020, 12, 3053 6 of 13 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.
Nutrients 2020, 12, x FOR PEER REVIEW 6 of 14 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.    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). 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 HFDtreated 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).
As a result of the measurement of fat content in the liver using Oil-Red-O staining, the HFDtreated 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).
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. 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).   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).   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. 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.

Discussion
In the present study, the effects of PM and PF extracts on digestive enzymes were assessed. In in vitro studies, PM extract and PF extract have increased dose-dependent inhibitory activity on αamylase (IC50 value: 6.13 mg/mL) and pancreatic lipase (IC50 value: 1.68 mg/mL), respectively. Notably, α-amylase, one of the digestive enzymes secreted from the pancreas and salivary glands, is involved in important biological processes such as the digestion of carbohydrates. α-Amylase inhibitors are well known to be effective in reducing postprandial hyperglycemia by slowing the digestion of carbohydrates and absorbing postprandial glucose. Reducing postprandial 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).

Discussion
In the present study, the effects of PM and PF extracts on digestive enzymes were assessed. In in vitro studies, PM extract and PF extract have increased dose-dependent inhibitory activity on αamylase (IC50 value: 6.13 mg/mL) and pancreatic lipase (IC50 value: 1.68 mg/mL), respectively. Notably, α-amylase, one of the digestive enzymes secreted from the pancreas and salivary glands, is involved in important biological processes such as the digestion of carbohydrates. α-Amylase inhibitors are well known to be effective in reducing postprandial hyperglycemia by slowing the digestion of carbohydrates and absorbing postprandial glucose. Reducing postprandial

Discussion
In the present study, the effects of PM and PF extracts on digestive enzymes were assessed. In in vitro studies, PM extract and PF extract have increased dose-dependent inhibitory activity on α-amylase (IC 50 value: 6.13 mg/mL) and pancreatic lipase (IC 50 value: 1.68 mg/mL), respectively. Notably, α-amylase, one of the digestive enzymes secreted from the pancreas and salivary glands, is involved in important biological processes such as the digestion of carbohydrates. α-Amylase inhibitors are well known to be effective in reducing postprandial hyperglycemia by slowing the digestion of carbohydrates and absorbing postprandial glucose. Reducing postprandial hyperglycemia prevents glucose uptake into adipose tissue, inhibiting the synthesis and accumulation of triacylglycerol [24,25].
On the other hand, pancreatic lipase is one of the most important enzymes for the digestion of dietary triacylglycerols. It is well known that dietary lipid is not directly absorbed from the intestines unless it has been subjected to the action of pancreatic lipase [24]. It has been clinically reported that a pancreatic lipase inhibitor, orlistat, prevented obesity and hyperlipidemia through the increment of fat excretion in the feces and the inhibition of pancreatic lipase. Based on this fact, the inhibition of these digestive enzymes is an important factor in the treatment of obesity [26,27].
We confirmed the anti-obesity effect of DKB-117 by way of digesting enzyme inhibition through in vivo testing. As a result of the test, DKB-117 extracts revealed effects of reducing weight and total fat in HFD-induced obese C57BL/6J mice. Micro-CT imaging was performed to quantify fat volume (total fat, abdominal fat, and subcutaneous fat). This method can readily discriminate between subcutaneous and abdominal fat [28]. Our results revealed that DKB-117 was able to reduce these fats. The in vivo test results demonstrate that DKB-117 administration regulates serum biochemical parameters (TCHO, TG, HDL, and LDL) in HFD-induced obese mice. These results are associated with an anti-hypercholesterolemic effect of PF [13,14]. In addition, the DKB-117 extracts increased TG and carbohydrate emissions in feces compared to those in the HFD group. It is expected that DKB-117 extracts promote the release of fat and carbohydrates due to the α-amylase inhibitory effect and the lipase inhibitory effect. Garcinia cambogia, which is currently used as a dietary supplement, is known to help reduce body fat by inhibiting the enzymes needed to synthesize carbohydrates into fat [29].
On the other hand, the DKB-117 extracts are expected to have an anti-obesity effect through a complex mechanism that promotes carbohydrate release through the inhibition of carbohydrate-degrading enzymes and which inhibits lipid absorption through lipase inhibition. It was observed that DKB-117 can effectively inhibit weight gain in animal experiments through a complex mechanism of inhibition of pancreatic lipase activity and inhibition of amylase activity, which is considered to be a suitable anti-obesity measure for Koreans who use carbohydrate as a staple food.
While the existing research on herbal extract (Garcinia cambogia, Plantago psyllium, Morus alba) single-action mechanisms is underway, it has been confirmed through this study that they are effective in anti-obesity in the battle against DKB-117 multifunction machines. Based on the results of in vitro and in vivo tests, the anti-obesity effects of DKB-117 in human application tests will be confirmed. Furthermore, it is determined that active ingredients should be investigated through the study on the separation of components of DKB-117.
In the future, the safety and efficacy of the DKB-117 extracts should be demonstrated through ongoing clinical trials; we plan to search for the activity compound in the DKB-117 extracts as well as perform additional testing of enzyme activity for lipid metabolism so that it can be widely used as a functional food material.

Conclusions
In summary, results of the present study revealed that DKB-117 extracts possess significant anti-obesity activities. These data clearly show that DKB-117 extracts are expected to have an anti-obesity effect through a complex mechanism that promotes carbohydrate release through the inhibition of carbohydrate-degrading enzymes while blocking lipid absorption through lipase inhibition.
Pancreatic lipase is an enzyme that is secreted from the pancreas and hydrolyzes the ester bonds of triglycerides to produce glycerol and fatty acids.
Decomposed glycerol and fatty acids are absorbed by the mucosal cells of the small intestine and are used as an energy source, but fat that has not been used as an energy source is synthesized into triacylglycerol again through the monoacylglycerol pathway and accumulated in the body.
Suppressing pancreatic lipase activity inhibits hydrolysis of triacylglycerol into glycerol and fatty acids, thus inhibiting liposuction through the small mucous membrane, reducing the amount accumulated in the body to prevent obesity.
By suppressing the a-amylase activity, the hydrolysis of ingested polysaccharides is inhibited, so that the absorption of excessively ingested carbohydrates in the body can be reduced and obesity can be prevented. It was confirmed that DKB-117 exhibits an anti-obesity effect by a method in which not only suppression of fat absorption but also suppression of excess carbohydrate absorption act together.
Based on the above results, our team plans to secure the anti-obesity effect and safety of DKB-117 extract through clinical trials.