**1. 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].

Currently, four weight-loss medicines (orlistat marketed as Xenical®, Roche Holding AG, Basel Switzerland or Alli®, GlaxoSmithKline, Brentford, UK; Contrave® from Nalpropion Pharmaceuticals, San Diego, CA, USA; Belviq ® from Eisai, Tokyo, Japan; and Qsymia ® from Vivus, Campbell, CA, USA) have been approved by the United States Food and Drug Administration, with users' body weight regulated by pancreatic lipase inhibition, increased energy consumption, and appetite suppression [5,6]. However, existing synthetic drugs have been reported to cause heart attack and stroke as well as liver damage [7].

Accordingly, there have been a number of studies conducted on the development of anti-obesity materials that are e ffective in decreasing appetite and reducing weight by using natural substances with long histories of use [8,9]. According to a recent report, many herbal extracts (*Garcinia cambogia*, *Plantago psyllium*, *Morus alba*) have been suggested to act on fat and carbohydrate metabolism to regulate body weight [10].

However, a lot of safety issues related to hepatic insu fficiency, hepatitis, and heart disease have been reported in the case of *Garcinia cambogia* extract, which is the most widely sold and health functional food material in the world. Accordingly, it is required that new natural materials with good safety and e fficacy be developed.

White kidney beans (*Phaseolus multiflorus* var. albus Bailey; PM) are native to Italy and belong to the Leguminosae family. Abdulwahid et al. reported that the white kidney bean–treated group in a diabetic-induced mouse model showed glucose and weight loss e ffects compared to the control group [10]. In addition, many studies on anti-obesity-related clinical trials have reported that white kidney bean ingestion is e ffective in weight loss, waist circumference reduction, and weight loss through α-amylase [11].

*Pleurotus eryngii* var. ferulae (PF) is a mushroom of the family Pleurotaceae that is rich in protein and dietary fiber [12]. Wang et al. and Alam et al. reported on the antioxidative, anti-inflammatory, and hypotensive e ffects of PF. In addition, PF water extracts reduced body weight, white adipose tissue weight, and liver weight in a mouse model of obesity induced by a high-fat diet (HFD) while improving glucose tolerance [1,13,14].

One of the most important strategies in the treatment of obesity includes the development of nutrient digestion and absorption inhibitors in an attempt to reduce the degree of energy intake through gastrointestinal mechanisms without altering any central mechanisms. The inhibition of digestive enzymes is one of the most widely studied mechanisms used to determine the potential e fficacy of natural products as anti-obesity agents [15].

This study was carried out to develop a dietary supplement ingredient that improves convenience of use and has good safety and obesity e ffects by respectively mixing natural plants having α-amylase inhibitory and pancreatic lipase inhibitory e ffects. In the literature, α-amylase inhibitors are well documented to be e ffective in reducing postprandial hyperglycemia by slowing the digestion of carbohydrates and absorbing postprandial glucose [10]. Reducing postprandial hyperglycemia prevents glucose uptake into adipose tissue to inhibit the synthesis and accumulation of triacylglycerol. Lipase is a hydrolytic enzyme from the pancreas that changes triglycerides (TGs) to glycerol and fatty acids. Thus, the inhibition of lipase has an important role in the treatment of obesity by inhibiting fat absorption. Previously reported studies have shown the α-amylase–inhibitory e ffect of PM and the pancreatic lipase–inhibitory e ffect of PF [12,13].

Research is actively underway to find e ffective anti-obesity drugs or anti-obesity health functions. The authors of this study want to confirm the anti-obesity e ffect by mixing *Phaseolus multiflorus* var. albus Bailey with α-amylase inhibitory e ffect and *Pleurotus eryngii* var. ferulae reported to have a pancreatic lipase inhibitory e ffect. In this study, the optimum mixing ratio of PF extract and PF extract was selected by an in vitro test, and we sought to determine the anti-obesity e ffect of DKB-117 in digestive enzyme inhibition in a mouse model of obesity induced by HFD.

#### **2. Materials and Methods**

#### *2.1. Plant Material Collection and Extract Preparation*

DKB-117, which is a mixture of PM extract and PF extract, was provided by Dongkook Pharm. Co., Ltd. (Suwon, Korea). The lot number was DKB-117. PM (*Phaseolus multiflorus* var. albus Bailey) used in this study was cultivated from Egypt and purchased through Solim trading Co., Ltd. in Korea. PF (*Pleurotus eryngii* var. ferulae) was obtained from the DDLE A CHE Co., Ltd. (Cheonan, Korea). A voucher specimen number (DK0117) has been deposited at the R&D Center, Dongkook Pharm. Co., Ltd.

PM and PF were cut into small pieces and extracted with a 5-fold volume of 30% ethanol (v/v) at 80 ◦C for 10 h, in order to obtain two ethanolic extracts, namely PM and PF, respectively. After extraction, each solution was concentrated on a rotary evaporator (BUCHI Labortechnik AG, Flawil, Switzerland) until constant weight, and the PM extract was dried by freeze-drying and the PF extract was dried using a spray dryer. DKB-117 was prepared by mixing the previously prepared PM extract and PF extract at a weight ratio of 3:1.

#### *2.2.* 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-bu ffered 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% I2/0.05 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).

#### *2.3. 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 bu ffer 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 bu ffer (100 mM of Tris-HCl and 5 mM of CaCl2; pH: 7.0). Then, 100 μL of DKB-117 or orlistat was mixed with 880 mL of the enzyme bu ffer 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 inhibition (%) = [1 − (B − b)/(A − a)] × 100

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.
