Compounds Contributing to the Modulation of Visceral Adiposity and Hepatic Lipid Metabolism in High-Fat-Diet-Fed Rats by Pometia pinnata (Matoa) Peel Powder: Identification of Pancreatic Lipase Inhibitors

Abstract

Background: Pometia pinnata (matoa) peel powder attenuates high-fat diet-induced adiposity and hepatic lipid accumulation in rats, but the responsible compounds remain unclear. This study aimed to identify the bioactive compounds that may contribute to this phenotype, with an emphasis on pancreatic lipase inhibition as a candidate mechanism. Methods: Rats received high-fat diets containing matoa peel powder, or its water- or ethanol extraction residue. Visceral fat accumulation, hepatic lipid deposition, and serum lipid profiles were evaluated. An ethanol extract was fractionated by activity-guided column chromatography based on pancreatic lipase-inhibitory activity, and structures were identified by nuclear magnetic resonance analysis. Static in vitro gastrointestinal digestion was performed to assess inhibition of fatty acid release by the extract or isolated compounds. Results: The visceral adiposity- and hepatic lipid-modulating effects observed with matoa peel powder were retained in the water extraction residue but not in the ethanol extraction residue, suggesting removal of bioactive constituents by ethanol extraction. The ethanol extract inhibited pancreatic lipase (IC₅₀ = 740 µg/mL). Two active compounds—hederagenin saponin and protocatechuic acid—were isolated, and both inhibited pancreatic lipase (IC₅₀ = 149 µmol/L and 404 µmol/L, respectively). Under simulated digestion in vitro, the ethanol extract and protocatechuic acid reduced free fatty acid release, whereas hederagenin saponin did not. Conclusions: Matoa peel powder contains ethanol-soluble constituents, including pancreatic lipase-inhibitory compounds that may contribute to the modulation of adiposity and hepatic lipid metabolism in high-fat-diet-fed rats. The attenuation of individual-compound activity under simulated digestion is consistent with matrix- and intestinal milieu-dependent effects, and supports a multi-component mechanism involving saponins, phenolics (protocatechuic acid), and their intestinal biotransformation products.

1. Introduction

The prevalence of obesity and overweight has been steadily increasing in recent years, making them significant health concerns globally. According to the World Health Organization (WHO), in 2022, worldwide, an estimated 890 million adults were obese (body mass index [BMI] ≥ 30 kg/m²) and 2.5 billion were overweight (BMI 25–29.9 kg/m²), representing approximately 16% and 43% of the global adult population, respectively [1]. The global obesity prevalence is approaching one billion [1]. If current trends persist, more than half of the world’s population will be overweight or obese by 2050 [2].

Obesity, a chronic condition, is caused by an imbalance between energy intake and expenditure. It is commonly associated with an increased risk of certain noncommunicable diseases (NCDs), including cardiovascular disease, type 2 diabetes, and certain cancers [1,3]. Treatment options for obesity include dietary interventions, physical activity, self-efficacy enhancement, pharmacotherapy, and bariatric surgery [4]. Lifestyle modification remains the cornerstone of obesity management and is essential even when pharmacological or surgical approaches are used [5,6]. Functional food components have recently attracted attention for their potential to support lifestyle improvements. In particular, phytochemical bioactive compounds such as polyphenols exert anti-obesity effects through multiple mechanisms.

Pometia pinnata, commonly known as matoa (Island Lychee or Fijian Longan), is a tropical fruit of the genus Pometia, family Sapindaceae [7]. It is widely distributed from South China and Sri Lanka through Southeast Asia to the Pacific Islands. In local markets, fresh fruit and roasted seeds are consumed, while extracts from the leaves and bark have traditionally been used to treat stomach upset, diarrhea, dysentery, colds, and flu [8]. Different parts of matoa—including leaves, fruit, bark, peel, and roots—exhibit diverse pharmacological properties, such as anti-bacterial, analgesic, anti-cancer, anti-diarrheal, anti-HIV, anti-hyperglycemic, anti-hypertensive, diuretic, and nephroprotective effects, along with a notable antioxidant activity [9]. In addition to these properties, its potential metabolic benefits have attracted research attention. Antioxidants in the fruit peel of matoa are reportedly higher than those in the fruit pulp or seeds [10].

We previously investigated the antioxidant activity and bioavailability of the peels and seeds of six tropical fruits from Indonesia [11]. Among them, the peels of matoa and salak exhibited high total phenolic content and strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity and lipid peroxidation inhibition. Based on these findings, we investigated the effects of matoa and salak peels on body composition and blood biomarkers in rats fed a high-fat diet. Matoa peel powder (MPP) supplementation significantly reduced body weight, visceral fat, serum triglyceride levels, and liver lipid content, but not salak peel powder (SPP) [12]. MPP contained over 90-fold higher hederagenin saponin than SPP; however, the specific compounds in matoa peel underlying these metabolic effects have not yet been identified. Saponins and polyphenols with antioxidant properties have been reported to exert inhibitory effects against pancreatic lipase [13,14]. While these natural compounds generally exhibit weaker pancreatic lipase inhibitory activity than pharmaceutical agents such as Orlistat, they may offer complementary dietary approaches for obesity management. Identifying compounds in MPP with adiposity- and hepatic lipid-modulating effects is crucial to elucidating their mechanism of action.

Therefore, this study aimed to identify bioactive compounds responsible for the beneficial metabolic effects of MPP, with a focus on pancreatic lipase inhibition. We first compared the in vivo effects of the residues remaining after water and ethanol extractions of MPP. The ethanol extraction residue lost the visceral adiposity- and hepatic lipid-modulating effects, indicating that the bioactive compounds were extracted into the ethanol solution. We therefore prioritized the ethanol extract for subsequent in vitro mechanistic analyses. The ethanol extract was then subjected to column chromatography guided by pancreatic lipase inhibitory activity, active constituents were isolated and characterized, and their effects were evaluated under static in vitro simulated gastrointestinal digestion conditions by quantifying free fatty acid release. This stepwise design was intended to separate the contributions of individual inhibitors from those arising from co-occurring components and potential biotransformation within the intestinal environment. To our knowledge, this is the first study to integrate residue-based in vivo comparison with activity-guided isolation from matoa peel and to evaluate inhibitor behavior under static in vitro simulated gastrointestinal digestion conditions.

2. Materials and Methods

2.1. Plant Material and Chemicals

2.1.1. MPP Preparation

Matoa was obtained from the local market in Bogor, Indonesia, in October 2020. MPP was prepared as described previously [12] and stored at 4 °C. The composition of MPP was analyzed following a previously described protocol [12].

2.1.2. Ethanol Extract of MPP (EX) and Its Extraction Residue

Ethanol (1000 mL) (Sigma-Aldrich, St. Louis, MO, USA) was added to MPP (182 g), heated to 60 °C, and stirred for 2 h. The mixture was cooled and allowed to stand for 18 h, and filtered through a non-woven fabric tea filter (Tokiwa Kogyo, Osaka, Japan) and a thin cotton flannel bag for broth (Yoshida Orimono, Niigata, Japan) to separate the filtrate from the residue. The residue was subjected to extraction twice with ethanol (1000 mL × 2). The collected filtrates were concentrated and dried under reduced pressure to obtain the crude EX (12.9 g) as an oily residue. The solid residues remaining after ethanol extraction were spread at 15 °C in the dark for 11 days and then dried in vacuo for 7 h to obtain the powder (131 g), referred to as the ethanol (alcohol) extraction residue (solid marc) of MPP (AR).

2.1.3. Water Extraction Residue of MPP

Ultrapure water (1000 mL) (PURELAB flex 3, ELGA, Wycombe, UK) was added to MPP (50 g × 2, packed in non-woven fabric filter bags) in a water-boiling pot (HMJ3-1000W, HARIO, Tokyo, Japan) and heated to approximately 100 °C for 3 h. The mixture was cooled, allowed to stand for 18 h, and filtered through a polyester fabric bag for broth (Yoshida Orimono, Niigata, Japan). The residue was subjected to extraction twice with ultrapure water (1000 mL × 2). The solid residues remaining after water extraction were spread at 15 °C in the dark for 7 days and subsequently dried in vacuo for 8 h to obtain a residual powder (72.5 g), referred to as the water extraction residue (solid marc) of MPP (WR). Whole water extraction was repeated to obtain an additional residue (74.0 g).

2.1.4. Chemical Analyses

The supernatant from the sonicated mixture of the EX (4.0 g) (Section 2.1.2) in ethyl acetate (200 mL) was separated and applied to a silica gel column for flash chromatography. The elution was performed using a gradient flow from 0% to 100% ethyl acetate in hexane to obtain fractions 1 to 9. Fraction 7 was eluted with 400 mL of 50% ethyl acetate/hexane and contained a crude oil (56 mg). The crude oil (4.3 mg) was purified with silica gel column chromatography with an isocratic eluent of hexane/acetone/acetic acid (30:25:1) to obtain compound II (2.3 mg), identified as protocatechuic acid by comparing its nuclear magnetic resonance (NMR) spectrum with that of authentic 3,4-dihydroxybenzoic acid (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan).

Specific data for compound II: ¹H NMR (600 MHz, (CD₃)₂SO referenced to 2.50 ppm) δ 6.76 (d, J = 8.4 Hz, 1H, H-5), 7.27 (dd, J = 2.4, 8.4 Hz, 1H, H-6), 7.32 (d, J = 1.8 Hz, 1H, H-2), 9.32 (br s, 1H, OH), 9.69 (br s, 1H, OH), 12.33 (br s, 1H, C(O)OH); ¹³C NMR (150 MHz, (CD₃)₂SO referenced to 39.50 ppm) δ 167.42 (C(O)OH), 150.02 (C-4), 144.91 (C-3), 121.90 (C-6), 121.75 (C-1), 116.57 (C-2), 115.17 (C-5); LRMS m/z 154 (M⁺).

Compound I (15.3 mg), hederagenin saponin, was obtained and chemically identified by NMR spectroscopy using the method described in the literature [12]. Unless otherwise stated, all solvents and silica gel used in this section, as well as inorganic salts and Tris used in the following sections, were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan).

2.2. Animal Experiments

The experimental protocol for the care and use of the animals was conducted in accordance with ethical standards approved by the Animal Care and Use Committee of Wayo Women’s University (Chiba, Japan; approval number: 2201; approved on 26 August 2022).

The sample size for this experiment was determined by power analysis [15]. The sample size was calculated based on the effect size (Cohen’s f = 0.77), depending on the amount of visceral fat accumulation in a previous study examining the effects of matoa, a significance level of 5%, a power of 80%, and a one-way analysis of variance with four experimental groups. Consequently, the sample size required for comparison between the four groups was 24 animals (6 animals per group).

Twenty-four male Sprague–Dawley rats (4-week-old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). The animals were acclimated for 2 weeks on a standard chow diet (CE-2, CLEA Japan, Inc., Tokyo, Japan) and housed in plastic cages (W280 × L440 × H200 mm; 2–3 rats per cage) with clean-tip bedding (CL-4161, CLEA Japan, Inc.). Environmental conditions were maintained at 25 ± 2 °C with 50–60% relative humidity and a 12 h light/dark cycle. Food and water were provided ad libitum.

After acclimation, the rats (average body weight: 246.7 ± 9.7 g) were individually housed in metabolic cages (W200 × L250 × H200 mm; KN-646-B2, Natsume Seisakusho Co., Ltd., Tokyo, Japan), randomly divided into four groups (n = 6 per group), and fed experimental diets for 4 weeks. Body weight was measured weekly.

Dietary groups were as follows:

1. HF group: High-fat diet (approximately 40% of calories from fat).

2. 3M group: High-fat diet supplemented with 3% MPP.

3. 3WR group: High-fat diet supplemented with 3% WR.

4. 3AR group: High-fat diet supplemented with 3% AR.

The basal composition of the experimental diets was based on the AIN-93G formulation (American Institute of Nutrition) [12,16]. The diet was provided in a powdered form. Among the diet ingredients, L-cystine, soybean oil, and t-butylhydroquinone were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan). All other diet ingredients were obtained from Oriental Yeast Co., Ltd. (Tokyo, Japan). Detailed diet compositions are shown in Table 1. Daily food intake was monitored by weighing the initial and remaining food over 24 h. Energy intake was calculated by subtracting the leftover food weight from the initial weight.

Table 1.

The composition of various controlled diets fed to Sprague-Dawley rats for 4 weeks.

Parameter	Diet Group
	High-Fat (HF)	HF + 3% MPP (3M)	HF + 3% WR (3WR)	HF + 3% AR (3AR)
Energy (kJ/kg)	19,500	19,500	19,500	19,500
Ratio of energy-producing nutrients (kJ%)
Protein	17	17	17	17
Carbohydrate	43	43	43	43
Fat	40	40	40	40
Ingredients (g/kg diet)
Bovine milk casein	200	200	200	200
L-cystine	3	3	3	3
Corn starch	262.5	262.5	262.5	262.5
α-Corn starch	132	132	132	132
Sucrose	102.5	102.5	102.5	102.5
Soybean oil	25	25	25	25
Lard	180	180	180	180
t-Butylhydroquinone	0.005	0.005	0.005	0.005
Cellulose	50	20	20	20
AIN-93G Mineral mix	35	35	35	35
AIN-93G Vitamin mix with choline bitartrate	10	10	10	10
Matoa peel powder	0	30	0	0
Water-extracted residue of MPP	0	0	30	0
Alcohol-extracted residue of MPP	0	0	0	30

MPP: matoa peel powder; WR: water extraction residue of MPP; AR: ethanol extraction residue of MPP.

At the end of the 4-week feeding experiment, the rats were anesthetized with isoflurane vapor following an overnight fast. Blood samples were collected from the abdominal aorta, and the animals were euthanized by exsanguination. Serum was separated by centrifugation at 1700× g for 10 min at 4 °C. The liver, kidneys, spleen, epididymal fat, perirenal fat, and mesenteric fat were excised and weighed. Serum and liver samples were frozen and stored at −80 °C until further analysis.

Serum biochemical analysis to determine serum glucose (Glc), triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT), aspartate transaminase (AST), and γ-glutamyl transpeptidase (γ-GTP) levels was performed using a DRI-CHEM 4000 chemistry analyzer (Fujifilm Holdings, Tokyo, Japan).

Hepatic lipid content was determined following a previously reported protocol [12]. In brief, total lipids were extracted from the liver samples using chloroform-methanol (2:1) [17], and the organic solvent was evaporated. The remaining lipid pellet was suspended in 2-propanol-Triton X-100 (9:1). TG and TC levels were determined using a Triglyceride E-Test (432-40201) and Cholesterol E-Test (439-17501), respectively, both from FUJIFILM Wako Pure Chemical Corp. (Osaka, Japan), following the manufacturer’s manual.

2.3. In Vitro Evaluation of the Pancreatic Lipase Inhibitory Activity of Extracts and Compounds

Pancreatic lipase inhibitory activity was determined using two methods:

Method A was based on the dimercaprol tributyrate–5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) colorimetric assay originally developed for serum lipase detection [18].

Method B was based on the quantification of free fatty acids released from triglyceride micelles digested by porcine pancreatic lipase. The protocol was modified from Morikawa et al. [19].

Orlistat (O0381, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was used as a positive control inhibitor, and 3,4-dihydroxybenzoic acid (protocatechuic acid) (C0055, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), which has the same structure as identified compound II, was used as a negative control inhibitor. All test samples, including those mentioned above, were dissolved in dimethyl sulfoxide (DMDO) (031-24051, Fujifilm Wako Pure Chemical Corp., Osaka, Japan).

2.3.1. Method A

Pancreatic lipase inhibitory activity was measured using Lipase Kit S (BS-92101, Sumitomo Bakelite Co., Ltd., Tokyo, Japan), including substrate solution containing dimercaprol tributyrate, an esterase inhibitor (phenylmethylsulfonyl fluoride), a coloring agent containing 5,5′-dithiobis(2-nitrobenzoic acid), and a stop solution. Porcine pancreatic lipase (100817, MP Biomedicals, LLC., Solon, OH, USA) was used as the enzyme source. For the assay, 73 µL of the coloring agent, 2 µL of the esterase inhibitor, and 5 µL of the lipase solution (150 µg/mL in 0.100 mol/L Tris-HCl, pH 8.0) were mixed and transferred to a 96-well microplate. The mixture was preincubated at 30 °C for 5 min. Next, 10 µL of test sample solution or DMSO (used as a control solvent) and 10 µL of substrate solution were added to each well, followed by incubation at 37 °C for 30 min. The reaction was terminated by adding 200 µL of stop solution. For the blank group, incubation at 37 °C was performed without the substrate solution, added only after the stop solution. Lipase activity was determined by measuring absorbance at 410 nm using a microplate reader (SH-1200Lab, Corona Electric Co., Ltd., Ibaraki, Japan). The inhibitory activity was expressed as the inhibition rate:

Inhibition rate (%) = 100 × (Abs_sample − Abs_sample,blank)/(Abs_control − Abs_{control,blank})

2.3.2. Method B

This assay comprised two steps: triglyceride digestion and free fatty acid quantification.

Triglyceride digestion was performed as follows. A substrate micelle suspension was prepared by sonicating a glass tube containing 40 mg triolein (T7140, Sigma-Aldrich, Saint Louis, MO, USA), 5.0 mg lecithin (120-00832, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), and 2.5 mg sodium taurocholate (191-10031, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) in 4.5 mL of 0.1 mol/L Tris-HCl (pH 7.0)/0.1 mol/L NaCl (Tris-HCl/NaCl buffer) for 30 min at 20–25 °C. Subsequently, 100 µL of the sonicated micelle suspension was transferred to a micro test tube and preincubated with 5 µL of test sample solution or DMSO and 95 µL of the Tris-HCl/NaCl buffer for 3 min at 37 °C. Subsequently, 50 µL of porcine pancreatic lipase (150 µg/mL in the Tris-HCl/NaCl buffer) or buffer alone (blank) was added, and the mixture was incubated at 37 °C for 40 min. The reaction was terminated by heating the tube at 95 °C for 2 min in an aluminum block incubator.

Free fatty acid concentration resulting from lipase activity was determined using LabAssay^TM NEFA (FFA) for cell biology (299-94301, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) following the manufacturer’s instructions. The relative amount of free fatty acids produced by triolein digestion was quantified by measuring absorbance at 550 nm. The inhibitory activity was expressed as the inhibition rate, calculated using the formula described in Section 2.3.1.

2.4. Evaluating the Inhibitory Effects of MPP, Extracts, and Compounds on Free Fatty Acid Release During Static In Vitro Digestion of a High-Fat Diet

Powdered high-fat diet was subjected to static in vitro simulated gastrointestinal digestion following the INFOGEST protocol described in Brodkorb et al. [20], with minor modifications.

Initially, 1.0 g of powdered high-fat diet—either alone or supplemented with MPP, extracts, or compounds—was suspended in 1.9 mL of simulated salivary fluid (pH 7.0), containing 15.1 mmol/L KCl, 3.7 mmol/L KH₂PO₄, 13.6 mmol/L NaHCO₃, 0.15 mmol/L MgCl₂, and 1.5 mmol/L CaCl₂ in a 15 mL conical tube. The mixture was agitated by orbital rotation at 99 rpm at 37 °C for 2 min. Subsequently, 2.9 mL of simulated gastric fluid (pH 3.0), containing 6.9 mmol/L KCl, 0.9 mmol/L KH₂PO₄, 25 mmol/L NaHCO₃, 47.2 mmol/L NaCl, 0.12 mmol/L MgCl₂, 0.15 mmol/L CaCl₂, and 2000 U/mL pepsin (P6887, Sigma-Aldrich, St. Louis, MO, USA), was added. The pH was adjusted to 3.0 with 5 mol/L HCl, and gastric digestion was performed by orbital rotation at 99 rpm and 37 °C for 2 h. Next, 5.8 mL of simulated interstitial fluid (pH 7.0), containing 6.8 mmol/L KCl, 0.8 mmol/L KH₂PO₄, 85 mmol/L NaHCO₃, 38.4 mmol/L NaCl, 0.33 mmol/L MgCl₂, 0.60 mmol/L CaCl₂, 1 mg/mL pancreatin (P7545, Sigma-Aldrich, St. Louis, MO, USA), and 12.5 mg/mL bile extract porcine (B8631, Sigma-Aldrich, St. Louis, MO, USA), was added. The pH was adjusted to 7.0 with 5 mol/L NaOH. Before initiating intestinal digestion, a 100 µL aliquot was transferred to a chilled, ice-filled tube to serve as a blank. Intestinal digestion was performed by orbital rotation at 80 rpm at 37 °C for 2 h. The digestion was terminated by adding 80 µL of 4-phenyl bromophenylboronic acid (B1858, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and the digest was immediately transferred to ice.

Quantification of free fatty acid concentrations was performed using LabAssay^TM NEFA (FFA) for cell biology, as described in Section 2.3.2. The inhibitory effects were expressed as the inhibition rates, calculated using the formula described in Section 2.3.1.

2.5. Statistical Analysis

A power analysis to determine the number of animals required for the experimentation was performed using G*power (version 3.1.9.7, G*Power Team, Heinrich Heine University Düsseldorf, Germany) [15].

Statistical analyses were performed using Microsoft Excel for Microsoft 365 (version 2510; Microsoft Corporation, Redmond, WA, USA). Normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test) were assessed using the Stats Kingdom web calculator (https://www.statskingdom.com/230var_levenes.html; accessed on 15 January 2026). Omnibus tests (e.g., ANOVA or Kruskal–Wallis, as appropriate) and multiple-comparison tests were performed using js-STAR XR+ (release 2.4.2 j; https://www.kisnet.or.jp/nappa/software/star/; accessed on 15 January 2026). Results are expressed as mean ± standard deviation. Before testing for differences among groups, we evaluated distributional assumptions. Normality within each group was assessed using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test. When assumptions were met, we performed one-way analysis of variance followed by Tukey’s post hoc test. For the hepatic lipid data, Levene’s test rejected homogeneity of variances; therefore, we used the Kruskal–Wallis test followed by the Steel–Dwass post hoc test. A p-value of <0.05 was considered statistically significant. IC₅₀ values were calculated from dose–response curves using a four-parameter logistic regression model [21] implemented in the web application “Four Parameter Logistic (4PL) Curve Calculator” (AAT Bioquest; https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator; accessed on 15 January 2026).

3. Results

3.1. WR Exerts Adiposity- and Hepatic Lipid-Modulating Effects in High-Fat-Diet-Fed Rats, but Not AR

We first evaluated whether the compounds responsible for the metabolic effects of MPP were water- or alcohol-soluble using an animal experiment in which animals were fed a high-fat diet supplemented with water- or alcohol-extracted residues of MPP. In the experiment, the HF group served as a negative control, and the 3M group (MPP) was included as a benchmark based on its previously demonstrated metabolic effects [12].

Table 2 shows the initial and final body weights (BW) of the 4-week intervention, average daily dietary intake, and organ and fat tissue weights after the intervention. The average dietary and energy intake did not differ among the four groups. BW and liver, kidney, and spleen tissue weights did not differ. Visceral fat weights in the 3M group tended to be lower than those in the HF group, although only perirenal fat weight differed significantly. A similar effect was also observed in the 3WR group, but not in the 3AR group. We reassessed visceral fat weights by comparing pretesticular, perirenal, and mesenteric fat, and the sum of the three visceral fat weights per 100 g BW (Figure 1). As shown previously [12], the addition of 3% MPP (3M) to the high-fat diet significantly reduced perirenal and total visceral fat weights compared with the control HF group, demonstrating reproducibility of the findings. The addition of 3% of WR to a high-fat diet (3WR group) tended to decrease visceral fat weight, although the effect was weaker than that with 3M. In contrast, no decrease in effect was observed when 3% of the AR residue was added to the high-fat diet (3AR group).

Table 2.

Body, tissue, and organ weights and dietary intake in Sprague–Dawley rats fed four different controlled diets for four weeks.

Parameter	Experimental Group
	HF	3M	3WR	3AR
Initial BW (g)	247 ± 12 a	246 ± 10 a	248 ± 7 a	245 ± 9 a
Final BW (g)	428 ± 15 a	418 ± 19 a	425 ± 13 a	434 ± 16 a
Dietary intake (g/day)	20.3 ± 0.7 a	19.7 ± 0.9 a	20.3 ± 0.7 a	20.4 ± 0.7 a
Energy intake (kJ/day)	395 ± 14 a	385 ± 17 a	395 ± 13 a	399 ± 13 a
Liver (g)	11.6 ± 1.1 a	12.0 ± 0.9 a	11.6 ± 1.0 a	12.0 ± 0.9 a
Kidney (g)	2.7 ± 0.1 a	2.9 ± 0.2 a	2.9 ± 0.1 a	2.8 ± 0.2 a
Spleen (g)	0.7 ± 0.1 a	0.9 ± 0.1 a	0.8 ± 0.1 a	0.8 ± 0.1 a
Epididymal fat (g)	7.6 ± 1.2 a	5.9 ± 0.8 a	6.7 ± 1.4 a	7.8 ± 1.2 a
Perirenal fat (g)	10.3 ± 2.2 a	5.5 ± 0.7 b	6.7 ± 1.6 bc	9.3 ± 2.3 ac
Mesenteric fat (g)	6.4 ± 1.5 ab	4.3 ± 0.4 a	5.9 ± 0.5 ab	7.0 ± 2.1 b

BW, bodyweight; HF, high-fat diet (n = 6); 3M, high-fat diet containing 3% matoa peel powder (MPP) (n = 6); 3WR, high-fat diet containing 3% water extraction residue of MPP (n = 6); 3AR, high-fat diet containing 3% ethanol extraction residue of MPP (n = 6). Data are presented as means ± standard deviations. Means in the same row with different superscript letters are significantly different among groups (p < 0.05).

Figure 1.

The suppressive effect of MPP on visceral fat accumulation was retained in WR but not in AR in the high-fat-diet-fed rats. Relative weights (g per 100 g BW) of epididymal (A), perirenal (B), mesenteric (C), and total visceral fat (D) after 4 weeks of feeding. The total visceral fat weight was calculated by summing the weights of epididymal, perirenal, and mesenteric fat. Data are shown as dot plots with means ± standard deviations. HF, high-fat diet (n = 6); 3M, high-fat diet containing 3% matoa peel powder (MPP) (n = 6); 3WR, high-fat diet containing 3% water extraction residue of MPP (n = 6); 3AR, high-fat diet containing 3% ethanol extraction residue of MPP (n = 6). Means with different letters show a significant difference (p < 0.05).

Serum biochemical parameters and hepatic lipid analysis revealed that WR retained lipid-modulating effects, whereas the AR did not. Serum TG levels were significantly lower in the 3M group compared with the control HF group (Table 3). In the 3WR group, the lowering effect was observed, but its magnitude was lower than in the 3M group and was statistically insignificant; it became weaker in the 3AR group. The inhibitory effect of MPP on hepatic TG and TC accumulation, accompanied by a high-fat diet, was retained entirely in the 3WR group (Figure 2). In contrast, the effect disappeared completely in the 3AR group. Hepatotoxicity was not observed by the addition of MPP and its water- and alcohol-extracted residue into a high-fat diet, as there was no increase in serum ALT, AST, or γ-GTP levels (Table 2).

Table 3.

Serum glucose and lipid levels and serum hepatic enzyme activities in rats fed four different controlled diets for four weeks.

Parameter	Experimental Group
	HF	3M	3WR	3AR
Glucose (mmol/L)	10.6 ± 2.7 a	9.8 ± 2.0 a	10.4 ± 2.3 a	10.9 ± 2.6 a
TG (mmol/L)	0.97 ± 0.24 a	0.60 ± 0.18 b	0.70 ± 0.22 ab	0.84 ± 0.17 ab
TC (mmol/L)	1.29 ± 0.15 a	1.37 ± 0.19 a	1.48 ± 0.17 a	1.25 ± 0.17 a
HDL-C (mmol/L)	0.82 ± 0.13 a	0.94 ± 0.17 a	1.01 ± 0.17 a	0.83 ± 0.19 a
ALT (U/L)	17.3 ± 3.2 a	15.2 ± 0.4 a	15.7 ± 2.0 a	17.5 ± 2.7 a
AST (U/L)	64.8 ± 22.7 a	57.3 ± 7.3 a	53.2 ± 6.6 a	66.3 ± 7.2 a
γ-GTP (U/L)	<1	<1	<1	<1

HF, high-fat diet (n = 6); 3M, high-fat diet containing 3% matoa peel powder (MPP)(n = 6); 3WR, high-fat diet containing 3% water extraction residue of MPP (n = 6); 3AR, high-fat diet containing 3% ethanol extraction residue of MPP (n = 6); TG, triacylglycerol; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; ALT, alanine transaminase; AST, aspartate transaminase; γ-GTP, gamma-glutamyl transpeptidase. Data are presented as means ± standard deviations. Means in the same row with different superscript letters are significantly different among groups (p < 0.05).

Figure 2.

The suppressive effect of hepatic lipid accumulation by MPP was retained in WR but not in AR in the high-fat-diet-fed rats. Hepatic triacylglycerol (TG) (A) and total cholesterol (TC) (B) (g per g liver) after 4 weeks of feeding. Data are shown as dot plots with means ± standard deviations. HF, high-fat diet (n = 6); 3M, high-fat diet containing 3% matoa peel powder (MPP) (n = 6); 3WR, high-fat diet containing 3% water extraction residue of MPP (n = 6); 3AR, high-fat diet containing 3% ethanol extraction residue of MPP (n = 6). Means with different letters show a significant difference (p < 0.05).

Thus, most compounds in MPP that exhibit adiposity- and hepatic lipid-modulating effects remain in WR, while they can be easily extracted with ethanol. Therefore, in subsequent experiments, we decided to search for bioactive compounds using EX as the starting material.

3.2. EX Inhibits Pancreatic Lipase When Micellized Triglyceride Is Used as a Substrate, but Not in a Water-Soluble Substrate

Natural compounds in fruits, vegetables, and herbs with anti-obesity effects often exhibit pancreatic lipase inhibitory activity. Hederagenin saponins from the pericarps of Sapindus rarak, included in matoa but not in salak peel, which has no anti-obesity effect, inhibit the pancreatic lipase activity [19]. So, we explored the active compounds with pancreatic lipase inhibitory activity in EX.

Several methods to detect pancreatic lipase activity are available. We used two methods: Method A, using dimercaprol tributyrate, a soluble substrate for pancreatic lipase, and Method B, using triolein in an artificial bile-like micelle as a substrate for pancreatic lipase (Figure 3). Orlistat, a potent pancreatic lipase inhibitor, is used to reduce visceral fat. It has been approved in Japan, the USA, and the EU. It shows similar inhibitory effects in both Method A and Method B. EX showed potent inhibitory activity in Method B but weak activity in Method A, suggesting that the compounds may inhibit pancreatic lipase activity by disrupting the interaction between the substrate and enzyme only in bile-like micellized hydrophobic substrates and not by directly interacting with the enzyme’s active center. Therefore, we conducted a subsequent pancreatic lipase inhibition assay using Method B to focus on the target compounds.

Figure 3.

EX inhibits pancreatic lipase activity when micelle-encapsulated triacylglycerol is the substrate, but not with a water-soluble artificial substrate. Pancreatic lipase assay in the absence or the presence of EX with a water-soluble substrate (dimercaprol tributyrate) (A) or micelle-encapsulated triacylglycerol (B). Data are expressed as 100% in the absence of inhibitors and presented as a bar graph with means ± standard deviations from triplicate measurements. EX, ethanol extract of matoa peel powder. Orlistat, a potent pancreatic lipase inhibitor, is the positive control. Means with different letters show a significant difference (p < 0.05).

3.3. Identification and Pancreatic Lipase Inhibitory Activity of Bioactive Compounds In Vitro

As described above, saponins with pancreatic lipase inhibitory activity are potent target compounds for anti-obesity research. Among these are hederagenin saponins. Therefore, EX was passed through a short column of DIAION HP-20 used as solid phase extraction (SPE) and eluted with water followed by methanol to provide water-eluted and methanol-eluted fractions. Figure 4A shows the pancreatic lipase inhibitory activity of EX (open circle, IC₅₀ = 0.766 mg/mL), water-eluted fraction (open triangle, IC₅₀ = 1.57 mg/mL), and methanol-eluted fraction (open square, IC₅₀ = 0.277 mg/mL), showing that the targeted compounds were concentrated in the methanol-eluted fraction, similar to that reported by Morikawa et al. [19].

Figure 4.

Dose-inhibition curves of eluted fractions via solid phase extraction (A) and purified compound I (B) in pancreatic lipase activity. For panel (A), symbols represent: open circle, ethanol extract of matoa peel powder (EX); open triangle, water-eluted fraction; open square, methanol-eluted fraction. For panel (B), solid circles represent compound I. Data are expressed as 100% in the absence of inhibitors and presented as means ± standard deviations from triplicate measurements.

The active methanol-eluted fraction included a saponin reported previously [12], and compound I, 3-O-[α-L-Arabinofuranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-hederagenin, as described in Materials and Methods (Section 2.1.4). Figure 4B shows the pancreatic lipase inhibitory activity of compound I. Its IC₅₀ for compound I was 0.131 mg/mL (149 µmol/L), well within the range reported by Morikawa et al. [19], where the IC₅₀ values ranged from 100 to 172 μM. The shapes of the dose-inhibition curves for EX (Figure 4A, open circle) and purified compound I (Figure 4B) resembled each other; however, those of the methanol-eluted fraction differed, suggesting the existence of another compound, other than hederagenin saponins, and its involvement in pancreatic lipase inhibition. The other compound might be relatively lipophilic because it was detected in the methanol-eluted fraction, not in the water-eluted fraction.

We attempted to isolate compounds with pancreatic lipase inhibitory activity but that were structurally distinct from saponins using normal-phase silica gel column chromatography. For preparing starting samples for column chromatography, EX was dissolved in ethyl acetate, and the insoluble precipitate, such as sugar, was removed. Ethyl acetate-soluble materials were subjected to silica gel chromatography and eluted with a stepwise gradient of ethyl acetate-hexane solution, yielding nine fractions. Figure 5A shows the pancreatic lipase inhibitory activity of the nine obtained fractions at a concentration of 400 µg/mL. Among them, fraction 7 (Fr. 7) demonstrated the most potent inhibitory activity (Figure 5A). IC₅₀ of Fr. 7 was 112 µg/mL (Figure 5B). This revealed the existence of compounds structurally distinct from saponins that inhibit pancreatic lipase.

Figure 5.

Pancreatic lipase activity for fractions obtained from the ethanol extract of matoa peel powder (EX) by normal-phase silica gel column chromatography (A), dose-inhibition curve of Fr. 7 from the column chromatography (B), and protocatechuic acid (C). Open circles represent Fr. 7, and solid circles represent protocatechuic acid. Data are expressed as 100% in the absence of inhibitors and presented as means ± standard deviations from triplicate measurements.

Fr. 7 was subjected to column chromatographic steps, resulting in the identification of compound II as protocatechuic acid, 3,4-dihydroxybenzoic acid, yielding 53% of the extract (Section 2.1.4). Because protocatechuic acid is commercially available, we used 3,4-dihydroxybenzoic acid (TCI, Tokyo, Japan) as compound II for the subsequent analysis. Figure 5C shows the inhibitory activity for pancreatic lipase of protocatechuic acid. Its IC₅₀ was 62.2 µg/mL (404 µmol/L), almost half that of Fr. 7, suggesting that Fr. 7 contained nearly half of protocatechuic acid. The estimation corresponds well with the yield mentioned above.

3.4. EX and Compound II Inhibit the Release of FFAs During Intestinal Digestion in the Static Digestion System In Vitro, but Not Compound I

To assess whether the isolated compounds exert adiposity- and hepatic lipid-modulating effects through pancreatic lipase inhibition, we investigated their impact on TG digestion to FFA in a high-fat diet meal using a static in vitro digestion system. First, we confirmed the inhibition by Orlistat (tetrahydrolipstatin as CAS name), a well-known potent pancreatic lipase inhibitor clinically used in the management of obesity [22] (Figure S1). Orlistat strongly inhibited the FFA release after intestinal digestion, as well as pancreatic lipase activity, regardless of the water-soluble or micellized substrate (Figure S1). IC₅₀ values of pancreatic lipase inhibition using water-soluble and micellized substrates, and of the FFA release were 12.9, 15.2, and 34.7 ng/mL (20.6, 30.6, and 70.1 nmol/L), respectively. Although the IC₅₀ value for FFA release during intestinal digestion was higher than that for inhibiting pancreatic lipase activity, the validity of the in vitro digestion system for evaluating the adiposity- and hepatic lipid-modulating effects of the compounds was demonstrated. Similar differences in inhibitory activity between the pancreatic lipase enzyme system and the in vitro digestion system have been previously reported [23,24].

Next, we examined the inhibitory effects of EX, its methanol-eluted fraction obtained by SPE, protocatechuic acid, and hederagenin saponin. Figure 6A shows the weak but significant inhibition by EX, the methanol-eluted fraction, and protocatechuic acid. The methanol-eluted fraction and protocatechuic acid had dose-dependent inhibitory activity, although the inhibitory rates were less than 50% even at the highest possible concentrations (Figure 6A,B). However, unexpectedly, hederagenin saponin showed no such activity; instead, it enhanced FFA release at 20 and 500 µg/mL (23 and 566 µmol/L), respectively (Figure 6C). Thus, the visceral adiposity- and hepatic lipid-modulating effects of MPP in vivo might not be explained solely by the pancreatic lipase-inhibitory activities of hederagenin saponin and protocatechuic acid but rather by the action of multiple compounds, in addition to these two identified pancreatic lipase inhibitors, at various steps beyond pancreatic lipase inhibition.

Figure 6.

Comparison of the inhibitory effects on FFA release by the ethanol extract of matoa peel powder (EX), its methanol-eluted fraction, and purified compounds (A) and dose–response curves of protocatechuic acid (B) and hederagenin saponin (C) during static in vitro intestinal digestion of a high-fat diet. FFA contents in the digesta were measured using LabAssay^TM NEFA (FFA) before and after intestinal digestion, and the amount of FFA generated during digestion was calculated by subtracting the pre-digestion value from the post-digestion value. Data are expressed as percentages relative to the control (100% in the absence of inhibitors) and presented as means ± standard deviations from triplicate measurements (hexaplicate for control samples). Means with different letters show a significant difference (p < 0.05).

4. Discussion

Our previous work [12] demonstrated that MPP reduces body weight and improves metabolic parameters in high-fat-diet-fed rats, suggesting the presence of bioactive compounds, such as compound I. In this study, we identified two molecules, hederagenin saponin (compound I) and protocatechuic acid (compound II), demonstrating pancreatic lipase inhibitory activity. These compounds were isolated from the lipophilic fractions of EX, as observed in an animal experiment where the AR did not retain the visceral adiposity- and hepatic lipid-modulating effects, unlike WR, which still exerted these effects on high-fat-diet-fed rats. However, single molecules of compounds I or II did not reproduce the FFA release inhibition of MPP in in vitro intestinal digestion using a high-fat diet, unlike Orlistat.

Triterpenoid saponins from various plant species exhibit pancreatic lipase inhibitory activities in vitro, blunt postprandial plasma TG elevation following oral lipid challenges in rodents, and mitigate HFD-induced obesity phenotypes, including reduced bodyweight gain, visceral adiposity, and hepatic lipid accumulation [19,25,26,27,28,29,30]. The reported IC₅₀ values for pancreatic lipase inhibition by the triterpenoid saponins generally ranged from 150–500 µmol/L (approximately 150~700 µg/mL, depending on molecular mass), comparable to our measurements in this study. In contrast, in rodent models, the effective oral doses reported for anti-obesity effects are typically around 1 g/kg/day for many saponins. Lower effective doses (approximately 0.1 g/kg/day) have been reported for protopanaxadiol-type ginsenosides, based on the methodological descriptions in the respective studies [29]. These in vivo dose requirements appear to require much higher levels of hederagenin saponin than those achievable from our MPP diet.

Quantitative considerations support this interpretation. The hederagenin saponin content in the 3% MPP-containing HFD was estimated to be 423 mg/kg diet from our calculation of the saponin contents in MPP determined in the previous study [12], yielding an approximate daily intake of ~35 mg/kg/day for a 250 g rat consuming 20 g feed/day—substantially below typical saponin doses reported to produce anti-obesity effects. Moreover, only fractions of dietary saponins are released, solubilized, and available within the intestinal lumen during digestion [31,32]. Thus, pancreatic lipase inhibition by hederagenin saponins alone is insufficient to explain the anti-obesity effects of MPP on rats, and bioactivities of other saponins and their metabolites, as well as the role of additional bioactive compounds, are likely to contribute.

Differences in the dose–inhibition curve shapes observed for EX and the methanol-eluted fraction from SPE (represented by open circles and open squares in Figure 4A) suggest the presence of other bioactive compounds contributing to MPP’s effects. Typically, the dose–inhibition curve for the pancreatic lipase-inhibitory activity of saponins exhibits a characteristic profile: a slight enhancement at low concentrations, followed by a sharp increase in inhibition over a narrow concentration range [28,30,33]. Compound I, included in the methanol-eluted fraction, also displayed a similar pattern (Figure 4B). Therefore, we hypothesize that MPP may contain a compound with pancreatic lipase-inhibitory activity that is less hydrophilic or more lipophilic than saponins. To investigate this possibility, we attempted to isolate such compounds from EX using normal phase chromatography to remove saponins. Compound II, protocatechuic acid, was among the active ingredients in MPP, exhibiting pancreatic lipase inhibition measured by Method B, with triolein in an artificial bile-like micelle as a substrate.

Protocatechuic acid, a phenolic compound, is widely found in edible plants, including those utilized in traditional medicine, as well as in fruits, nuts, vegetables, and cereal grains, with particularly high concentrations in the outer layers, such as the bran and peel [34,35]. It can be generated from polyphenols metabolized by gut microbiota during colonic fermentation [34,36,37]. It exerts beneficial effects on serum and hepatic lipid profiles in animal models of metabolic dysfunction-associated steatotic liver disease (MASLD) at doses of 50–200 mg/kg/d by inhibiting pancreatic lipase activity, activating the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, and downregulating peroxisome proliferator-activated receptor γ (PPARγ) signaling [38,39,40,41]. Notably, the previously reported IC₅₀ for the in vitro pancreatic lipase inhibitory activity of protocatechuic acid (1.2–1.5 mg/mL) was markedly higher than our estimate (62.2 μg/mL) [41]. This discrepancy can be explained by the differences in assay conditions: we used triolein within an artificial bile-like micelle, whereas Li et al. utilized 4-methylumbelliferyl oleate in aqueous solution, with substantially different enzyme kinetics, substrate accessibility, and micellar partitioning.

EX contained compound I (saponin) at a weight that was 2.4 times larger than that of protocatechuic acid, as calculated by isolation yields and ¹H NMR measurement, estimated as protocatechuic acid at ≥0.1% of MPP. If this estimation holds, the amount of protocatechuic acid in a high-fat diet containing 3% MPP would be approximately 30 mg/kg diet, corresponding to a daily intake of about 2.5 mg/kg body weight (assuming a 250 g rat consumes 20 g of feed per day), substantially lower than the effective dose reported above. These calculations reinforce the view that additional constituents in EX—beyond hederagenin saponin and protocatechuic acid—likely participate in the anti-obesity activity and may function synergistically in multiple mechanisms in addition to pancreatic lipase inhibition. Tan et al. reported that the ethanol extract of Panax notoginseng exerted a greater effect on anti-obesity activity than the same dose amount of Panax notoginseng-derived saponin mixtures in high-fat-diet-fed mice [42].

We further propose multi-component and multi-mechanism actions. MPP constituents and their microbial metabolites (e.g., saponin secondary glycosides and aglycones) could act synergistically to (i) inhibit pancreatic lipase, (ii) modulate intestinal fatty-acid absorption and chylomicron assembly, and (iii) regulate hepatic lipid accumulation and oxidative stress, ultimately resulting in the observed visceral adiposity- and hepatic lipid-modulating effects of MPP. This conceptual model is consistent with reviews highlighting the potential roles of saponin metabolites generated by the gut microbiota in metabolic syndrome [43] and reports that certain aglycone-type triterpenoids, including hederagenin, exert anti-lipidemic effects in HFD-fed rodent models [44,45,46]. These are consistent with our previous finding that EX could suppress fatty acid-induced chylomicron production in intestinal epithelial cells [12]. Thus, a holistic mechanism in which MPP’s lipid-modulating effects emerge from complementary interactions among saponins, phenolics such as protocatechuic acid, and their biotransformation products within the intestinal milieu, rather than a simple mechanism that may influence TG absorption through pancreatic lipase inhibition, may be operational.

This study has some limitations that warrant further consideration. First, this study did not include a non-obese control group, which limits the interpretation of obesity-specific effects. However, our previous study included a normal-diet group (approximately 10% of calories from fat) alongside the HF and 3M groups [12], providing a non-obese baseline. The present study was specifically designed to identify compounds in MPP that modulate visceral and hepatic fat accumulation under high-fat dietary conditions. Second, in animal experiments, we used WR and AR to obtain bioactive extracts with visceral adiposity- and hepatic lipid-modulating effects, thereby identifying the active ingredients. This approach introduced uncertainty in intake assessment because the concentration of protocatechuic acid in MPP differs from that in EX, and no direct comparison was possible. Third, the current study employed a single dose (3% MPP) and a 4-week intervention. While this design was appropriate for bioactive compound identification, future work should examine the dose-response relationships of isolated compounds over longer periods using comprehensive metabolic profiling, including glucose and insulin assessments. Fourth, the intake amounts of the MPP-derived hederagenin saponin and protocatechuic acid in the rat model were lower than the effective doses of the purified compounds reported in the literature, which may not fully account for the observed correlation with physiological effects. Furthermore, the intake estimates were based on dietary intake and did not account for digestive absorption rates. Fifth, although the adiposity- and hepatic lipid-modulating mechanisms are hypothesized to involve pathways beyond pancreatic lipase inhibition—which may play a limited role—(e.g., fatty acid absorption inhibition or hepatic lipid metabolism regulation), solid mechanistic evidence was not obtained. In particular, key metabolic readouts—such as circulating free fatty acids, insulin or C-peptide, in vivo circulating pancreatic lipase activity, and hepatic expression of lipogenic genes—were not assessed, and a triglyceride tolerance test was not performed. Furthermore, the synergistic effects of multiple components and the contributions of metabolites produced by intestinal bacteria remain hypothetical and merit further experimental verification. Future studies integrating these metabolic assessments with metabolomic, transcriptomic, and microbiome analyses will enable a more comprehensive elucidation of the underlying mechanisms.

5. Conclusions

This study explored the compounds in the EX contributing to its visceral adiposity- and hepatic lipid-modulating effects, with a focus on pancreatic lipase inhibitory activity in vitro. We identified two compounds as active constituents: hederagenin saponin and protocatechuic acid. Although both compounds demonstrated lipase inhibition in vitro, their inhibitory effects on free fatty acid production under static in vitro simulated gastrointestinal digestion conditions were limited. These findings suggest that the adiposity- and hepatic lipid-modulating effects of MPP are mediated by a holistic mechanism involving complementary interactions among saponins, phenolics such as protocatechuic acid, and their biotransformation products within the intestinal milieu. Ongoing work by our group aims to further identify potent bioactive compounds from the fruit peel of Pometia pinnata and elucidate the mechanisms underlying these metabolic effects.

Abbreviations

The following abbreviations are used in this manuscript:

MPP	matoa peel powder
SPP	salak peel powder
EX	ethanol extract of matoa peel powder
AR	ethanol (alcohol) extraction residue of matoa peel powder
WR	water extraction residue of matoa peel powder
NMR	nuclear magnetic resonance
BMI	body mass index
DMSO	dimethyl sulfoxide
DPPH	1,1-diphenyl-2-picrylhydrazyl
Glc	glucose
TG	triacylglycerol
TC	total cholesterol
HDL-C	high-density lipoprotein cholesterol
ALT	alanine transaminase
AST	aspartate transaminase
γ-GTP	γ-glutamyl transpeptidase
BW	body weights
SPE	solid phase extraction
PPARγ	peroxisome proliferator-activated receptor γ
NRF2	nuclear factor erythroid 2-related factor 2

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/nu18050786/s1. Figure S1: Dose–response curves of Orlistat on in vitro pancreatic lipase activity using water-soluble substrate (A) and micellized substrate (B), and on FFA release during static in vitro intestinal digestion of a high-fat diet (C).

Author Contributions

Conceptualization, N.K. and T.S. (Toshikazu Suzuki); Methodology, N.K. and T.S. (Toshikazu Suzuki); Validation, N.K. and T.S. (Toshikazu Suzuki); Formal Analysis, A.T. (Ayumi Tago), N.K. and T.S. (Takahiro Sakai); Investigation, A.T. (Ayumi Tago), N.K., T.S. (Takahiro Sakai), A.T. (Ao Tian), S.T. and T.S. (Toshikazu Suzuki); Resources, N.K., T.S. (Takahiro Sakai), A.T. (Ao Tian), S.T., N. and J.N.; Data Curation, N.K. and T.S. (Toshikazu Suzuki); Writing—Original Draft Preparation, A.T. (Ayumi Tago), N.K. and T.S. (Toshikazu Suzuki); Writing—Review & Editing, All Authors; Visualization, A.T. (Ayumi Tago), N.K. and T.S. (Toshikazu Suzuki); Supervision, N.K. and T.S. (Toshikazu Suzuki); Project Administration, N.K. and T.S. (Toshikazu Suzuki); Funding Acquisition, N.K., J.N. and T.S. (Toshikazu Suzuki). All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The experimental protocol for the care and use of animals was approved by the Animal Care and Use Committee of Wayo Women’s University (Ethical approval number 2201; approved on 26 August 2022), and the experiment was conducted in accordance with the approved ethical guidelines.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Funding Statement

This study was funded by the Wayo Women’s University Fund for Research [T.S. (Toshikazu Suzuki)].