Isolation of Anti-Diabetic Active Compounds from Benincasae Exocarpium and Development of Simultaneous Analysis by HPLC-PDA

Diabetes is a chronic metabolic disease that is a constant problem. Previous studies have reported that Benincasa cerifera Savi. extracts are effective in treating diabetes and its complications. Benincasae Exocarpium (BE) is a fruit peel of B. cerifera that has been reported to be used for the prevention and treatment of metabolic diseases such as hyperglycemia, obesity, and hyperlipidemia. However, there are not enough studies on the compounds and bioassays to support the efficacy of BE. The inhibitory activity of the BE extracts and fractions against advanced glycation end-products (AGE) formation and α-glucosidase activity was evaluated. These assays are relevant for the treatment of type 2 diabetes and its complications. Based on these results, compounds 1–11 were isolated through bioassay-guided isolation. In addition, we developed a high-performance liquid chromatography (HPLC) method that can simultaneously analyze these 11 compounds. Activity evaluation of the compounds was also conducted, and eight compounds exhibited significant activity. Among these, flavonoid compounds showed strong activity. A quantitative evaluation of eight bioactive compounds (2, 5–11) was conducted. In conclusion, this study demonstrated the potential of BE for prevention and treatment of type 2 diabetes and its complications.


Introduction
The number of diabetic patients is steadily increasing worldwide, and type 2 diabetes, especially among young people such as children and adolescents, is becoming a problem [1]. The causes of type 2 diabetes include environmental exposure and genetic factors [2]. Early diagnosis and management of risk factors are important because diabetes causes various serious complications. Blood sugar management is important for the prevention and management of type 2 diabetes [3], and agents with α-glucosidase inhibitory activity are used as oral hypoglycemic drugs [4]. In the state of hyperglycemia, a sugar-derived substances called advanced glycation end-products (AGEs) are actively produced and accumulate in the blood and tissue [5,6]. AGEs are a heterogeneous group of molecules formed by the Maillard reaction [7]. The reaction is a non-enzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids [5]. Methylglyoxal (MGO) is one of the most reactive AGE precursors [8]. During aging and diabetes, increasing amounts of AGE-modified proteins can be detected. In other words, they are involved in the development of degenerative diseases such as diabetes [9]. Therefore, controlling the formation of AGEs is important for the prevention and treatment of diabetes and diabetic complications. Agents that inhibit or reduce AGE formation include aminoguanidine, pyridoxamine, and OPB-9195. Aminoguanidine has been reported to be toxic during clinical evaluation [6]. Therefore, it is necessary to identify a safe anti-glycation agent. To find novel synthetic AGE

Inhibitory Activity of the Extract and Fractions from Benincasae Exocarpium against AGE Formation in BSA-Glucose and BSA-MGO Systems
Advanced glycation end-products (AGEs) produced by hyperglycemia and oxidative stress are major causes of diabetic complications [5,34]. This is because AGEs cause structural and functional changes in proteins [35]. Therefore, inhibition of AGE formation is attracting attention in relation to the prevention of diabetic complications and the development of therapeutic agents [6]. To prove the potential of BE for preventing diabetic complications, we measured the AGE formation inhibitory activity in two systems: bovine serum albumin (BSA)-glucose and BSA-methylglyoxal (MGO). The results are shown in Table 1. In the BSA-glucose system, the extract, EA, and BuOH fractions from BE significantly inhibited AGE formation (IC 50 values of 684.12 ± 2.82, 628.05 ± 1.73, and 419.31 ± 1.28 µg/mL, respectively). In the BSA-MGO system, the EA and BuOH fractions were the most potent AGE formation inhibitors (IC 50 values of 214.95 ± 6.38 and 533.43 ± 16.04 µg/mL, respectively). The extract mildly inhibited AGE formation in BSA-MGO (IC 50 values of 842.38 ± 9.07 µg/mL). On the other hand, the Hx and water fractions showed no or slight inhibition of AGE formation in the two systems. Table 1. IC 50 (inhibitory activity) of the Benicasae Exocarpium (BE) extract and fractions against advanced glycation end-products (AGEs) formation in bovine serum albumin (BSA)-glucose and BSA-methylglyoxal (MGO) systems and α-glucosidase. Data are expressed as the mean ± SD (n = 3); a IC 50 was calculated from the least-squares regression line of the logarithmic concentrations plotted against the residual activity; b aminoguanidine hydrochloride (AMG) was used as a positive control of AGE formation inhibitory activities; c Acarbose was used as a positive control for α-glucosidase inhibitory activity; d ND: not detected; * indicates a significant difference from control; * p < 0.05, ** p < 0.005, *** p < 0.001; -: not measured.

Inhibitory Activity of the Extract and Fractions from Benincasae Exocarpium against A-Glucosidase
α-Glucosidase is a carbohydrase that catalyzes the liberation of α-glucose from the non-reducing end of the substrate [36]. Its inhibitors delay the absorption of carbohydrates and thus have a lowering effect on postprandial blood glucose and insulin levels [4].
Molecules 2022, 27, 9 5 of 14 Therefore, they are used in the treatment of type 2 diabetes. We assessed the potential involvement of the inhibition of α-glucosidase by BE, and the results are summarized in Table 1. The EA fraction was found to be most potent in the α-glucosidase inhibitory activity (IC 50 values of 212.20 ± 4.15 µg/mL). The Hx, BuOH, and water fractions mildly inhibited α-glucosidase (IC 50 values of 413.38 ± 3.58, 368.72 ± 9.24, 491.18 ± 12.67 µg/mL, respectively. In contrast, the extracts showed no inhibitory activity.

Development and Validation of Simultaneous HPLC-PDA Analysis
For the simultaneous analysis of 11 compounds isolated from BE, the HPLC-PDA method was developed. Among the 11 compounds to be tested, the absorption of each compound was recorded at different wavelengths: p-hydroxybenzoic acid (255 nm), protocatechuic acid (260 nm), isovanillin (231 nm), 5-hydroxymethylfurfural (282 nm), isovitexin (334 nm), vitexin (268 nm), orientin (226 nm), gallic acid (213 nm), caffeic acid (325 nm), vitexin-2 -O-rhamnoside (268 nm), and vitexin-4 -O-glucoside (268 nm). The maximum absorption wavelength of each compound and the peak area at 280 nm were compared and it was confirmed that there was no significant difference between the two cases. Therefore, the detection wavelength was determined to be 280 nm. Subsequently, an analysis method validation was performed. The specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision, and accuracy of the analysis method were confirmed. Additionally, the recovery and robustness were also confirmed (see Supplementary Materials, Tables S1 and S2) 2.6.1. Specificity By comparing a mixture of 11 compounds with an extract from BE, these were well separated without interference (Figure 1).

Development and Validation of Simultaneous HPLC-PDA Analysis
For the simultaneous analysis of 11 compounds isolated from BE, the HPLC-PDA method was developed. Among the 11 compounds to be tested, the absorption of each compound was recorded at different wavelengths: p-hydroxybenzoic acid (255 nm), protocatechuic acid (260 nm), isovanillin (231 nm), 5-hydroxymethylfurfural (282 nm), isovitexin (334 nm), vitexin (268 nm), orientin (226 nm), gallic acid (213 nm), caffeic acid (325 nm), vitexin-2″-O-rhamnoside (268 nm), and vitexin-4″-O-glucoside (268 nm). The maximum absorption wavelength of each compound and the peak area at 280 nm were compared and it was confirmed that there was no significant difference between the two cases. Therefore, the detection wavelength was determined to be 280 nm. Subsequently, an analysis method validation was performed. The specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision, and accuracy of the analysis method were confirmed. Additionally, the recovery and robustness were also confirmed (see Supplementary Materials, Tables S1 and S2) 2.6.1. Specificity By comparing a mixture of 11 compounds with an extract from BE, these were well separated without interference (

Linearity
The linearity of the 11 compounds was measured at five concentrations between 0.5-50 µg/mL. In all calibration curves, the correlation coefficient (r 2 ) of all compounds was greater than 0.999, and the results are shown in Table 3. Each value was presented by calculating the mean of triplication; a Y = peak area, x = concentration of standard (µg/mL); b r 2 = correlation coefficient for five final concentrations in the calibration curve; LOD, limit of detection; LOQ, limit of quantification.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD values of 11 compounds ranged from 0.05 to 2.92 µg/mL and LOQ values of all compounds ranged from 0.14 to 8.85 µg/mL (Table 3).

Intra-Day and Inter-Day Precision and Accuracy
Precision and accuracy were determined using 11 compounds mixture solutions. The results are summarized in Table 4. The intra-day and inter-day precisions of all compounds ranged from 0.14% to 5.69% and from 0.12% to 6.19%, respectively. The intra-day and inter-day accuracies of all compounds ranged from 92.1% to 108.6% and from 90% to 102.8%, respectively.

Quantitative HPLC-PDA Analysis of Isolated Compounds
As a result of screening the collected three samples of BE, 11 compounds were identified. Compounds 5 and 10 were identified as the major components of the BE. However, there was a compound with a large difference in the content of the components depending on the type of sample. Therefore, eight (2, 5-11) out of the 11 compounds were selected based on the activity assay. The eight bioactive compounds showed strong activity in the anti-glycation (AGE formation inhibitory) and anti-diabetic (α-glucosidase inhibitory) assays. HPLC-PDA analysis of the BE extracts was performed to quantitatively evaluate the bioactive compounds (Table 5). To optimize the extraction efficiency of these compounds, BE samples underwent extraction by altering the extraction solvent, (methanol [MeOH] and ethanol [EtOH]), the solvent ratios (30, 50, 70, and 100%), and the time (30, 60, 90, and 120 min). The sum of the peak areas of eight compounds was compared. Among the various extraction conditions, extraction done with 70% EtOH and during 60 min produced samples that contained the most eight marker compounds. Table 4. Intra-day and inter-day precision and accuracy of compounds 1-11.

Extraction, Fractionation, and Isolation of Benincasae Exocarpium
The BE (4.0 kg) was dried, powdered, and then extracted in methanol (20 L × 3) at room temperature. The filtrate was concentrated to dryness (406.03 g) in vacuo, suspended in water (H 2 O), and then partitioned using Hx, EtOAc, and BuOH. The results yielded Hx (100.46 g), EtOAc (21.15 g), BuOH (35.74 g), and water (154.22 g) fractions. Among these four fractions, the EtOAc and BuOH fractions were the most potent in the three anti-diabetes activity assays. In addition, in the fraction analysis using HPLC-PDA, various polar components were confirmed in the two fractions. Therefore, repeated open column chromatography of both active fractions was performed.

ESI-QTOF-MS/MS Analysis
The molecular weights of the isolated compounds 1-11 were determined using ESI-QTOF-MS/MS. Isolated compounds (1 mg) were dissolved in 10 mL of MeOH and filtered using a 0.45 µm syringe filter. The prepared compounds were directly injected into a Bruker Compact system.
The mobile phase was run on a gradient schedule with solvent A (water, 0.1% formic acid, v/v) and solvent B (acetonitrile, 0.1% formic acid, v/v). Solvent B was increased from 10% to 50% for 5 min and then from 50% to 100% for 10 min, maintained for 3 min, decreased from 100% to 10%, and maintained for 5 min. The flow rate of the mobile phase was 0.2 mL/min, and the injection volume was 30 µL. The optimal ESI-QTOF-MS/MS analysis conditions were as follows: source type electrospray ionization, ion polaritynegative, scan-50-800 m/z, set capillary-4500 V, set endplate offset-500 V, set nebulizer-1.2 bar, set dry heater-200 • C, and set dry gas-10.0 L/min.

HPLC-PDA Analysis
To analyze the isolated compounds (1-11), a Fortis C 18 Column (4.6 × 250 mm, 5 µm, Waters, Milford, MA, USA) was used. Mobile phases A and B consisted of water containing 1% acetic acid and acetonitrile, respectively, and were run on the following gradient schedule. Mobile phase A was maintained for 24 min and increased from 10% to 27%. The flow rate of the mobile phase was 1.0 mL/min, and the injection volume was 10 µL. Column temperature was maintained at 25 • C. Detection was performed at a UV absorbance wavelength of 280 nm. This analysis method is validated by various evaluation sections including specificity, linearity, LOD and LOQ, and intra-and inter-day precision and accuracy [37]. The samples were prepared as follows. Powdered BE samples (1 g) were dissolved in different solvents (MeOH: 30%, 50%, 70%, and 100%; EtOH: 30%, 50%, 70%, and 100%) and sonicated for different times (30,60,90, and 120 min). In addition, each sample was filtered using filter paper, and the solvent was removed in vacuo. The dried samples were dissolved in 1 mL of MeOH, filtered through a 0.45 µm syringe filter, and used as a sample solution. 1 mg of isolated compounds 1-11 were dissolved in 1 mL of MeOH and filtered through a 0.45 µm syringe filter. The compounds (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) were mixed in the same ratio to make a standard solution. They were diluted to concentrations between 2.5-50 ug/mL and used for HPLC-PDA analysis to validate the analysis conditions. 3.6. Bioactivity Assays 3.6.1. Inhibitory Activity against AGE Formation in BSA-Glucose System The AGE formation inhibitory assay of BSA-glucose was conducted using a spectrophotometric method developed in a previous study [38]. All test samples were dissolved in 10% DMSO at five different concentrations (1000-10,000 µg/mL for the extract and fractions and 100-10,000 µM for the compounds). The assay mixtures contained the following constituents: 135 µL of 50 mM phosphate buffer (pH 7.4) containing 0.02% sodium azide and BSA (10 mg/mL), 135 µL of 0.4 M D-fructose and D-glucose, and 30 µL of sample. The mixture was incubated at 60 • C for 2 days. After incubation, the fluorescence was measured at an excitation wavelength of 350 nm and emission wavelength of 450 nm in a 96-black well plate. AMG was used as a positive control. The inhibitory activity on AGE formation of BSA-glucose was calculated using the following formula: {(Ac − As)/Ac} × 100, where Ac is the fluorescence of the control and As is the fluorescence of the sample. Controls contained the same reaction mixture, except that phosphate buffer was added instead of the sample. The half-maximal inhibitory concentration (IC 50 ) values of triplicate measurements of the samples and AMG were calculated and expressed as the mean ± SD.

Inhibitory Activity against AGE Formation in BSA-MGO System
The AGE formation inhibitory assay of BSA-MGO was conducted using spectrophotometry according to a previously described method with modification [38,39]. All samples were dissolved in 10% DMSO. The assay mixtures contained 50 µL of 50 mM phosphate buffer (pH 7.4) containing 0.02% sodium azide and BSA (10 mg/mL), 50 µL of 7 mM MGO, and 50 µL of sample. After incubation at 60 • C for 2 days, fluorescence was measured on excitation and emission wavelengths of 340 and 420 nm, respectively, in a 96-black well plate. AMG was used as a positive control. The inhibitory activity on AGE formation by BSA-MGO was calculated using the same equation applied in the BSA-glucose assay.
3.6.3. Inhibitory Activity against α-Glucosidase α-Glucosidase inhibitory activity was measured spectrophotometrically according to the previously described method [40]. All samples were dissolved in 10% DMSO. This assay consisted of a 50 µL sample solution, 50 µL of 50 mM potassium phosphate buffer (pH 6.8), and 50 µL of 0.5 U/mL α-glucosidase was preincubated for 10 min at 37 • C. After incubation, 50 µL of p-NPG was added to the mixed assay mixtures. The reaction was performed in a 96-well plate, and the activity was measured using a spectrophotometer at 405 nm with acarbose as a positive control. The inhibitory activity was calculated using the equation applied in the BSA-glucose assay, but Ac and As are the absorbance values of the control and sample, respectively.

Statistical Analysis
Statistical significance was measured using one-way analysis of variance (ANOVA) and multiple comparisons with p < 0.05, p < 0.01, and p < 0.001 indicated statistical significance.

Conclusions
In this study, we confirmed the inhibitory activity of extracts and fractions of BE against AGE formation and α-glucosidase. Since EA and BuOH fractions exhibited the strongest inhibitory activities, we isolated 11 compounds (seven from EA fractions and four from BuOH fractions) in accordance with bioassay-guided isolation. They were identified by 1 H, 13 C-NMR, and ESI-QTOF-MS/MS. AGE formation and α-glucosidase inhibitory activity were confirmed for compounds 1-11. Among the isolated compounds, five flavonoids (5, 6, 7, 10, and 11) showed stronger inhibitory activity against AGE formation in BSAglucose and BSA-MGO systems than in positive controls. In addition, these compounds showed similar or high α-glucosidase inhibitory activity to the positive control group. Compound 2 showed high inhibitory activity against alpha-glucosidase and significant inhibition of AGE formation in BSA-MGO and BSA-glucose systems. Compounds 8 and 9 also showed significant inhibition of AGE formation in the BSA-glucose system. Compounds 1, 3, and 4 showed no or slight inhibitory activity. HPLC-PDA simultaneous analysis was developed for separated compounds 1-11, and validation was performed. Using the developed analysis method, the content of eight bioactive compounds (2 and 5) among compounds 1-11 was confirmed. The optimized extraction conditions for the eight compounds were 70% EtOH and 60 min of extraction. In addition, by presenting content standards for eight compounds, standards for quality management were prepared.
In summary, a systematic experiment (activity assays, compound isolation, and development of a multi-compound simultaneous content analysis method) was conducted to confirm that BE has an anti-diabetic effect. We confirmed anti-diabetic activity of the BE extract and its fractions. The various compounds were separated by open column chromatography, and anti-diabetic activity assays of the active ingredients were performed. In addition, the content standard of the BE was set through a multi-component simultaneous content analysis method using HPLC-PDA.
Based on these results, the possibility of BE as a functional material for the treatment of type 2 diabetes and diabetes complications was confirmed. Additional studies, including in vivo studies of BE, need to be conducted to evaluate whether efficacy is sufficient for clinical application and the drug effects of isolated compounds. Furthermore, compounds isolated from BE may be valuable functional agents against other diseases.