Triterpenoids from Kochiae Fructus: Glucose Uptake in 3T3-L1 Adipocytes and α-Glucosidase Inhibition, In Silico Molecular Docking

In this study, three new triterpenes (1–3) and fourteen known triterpenoids (4–17) were isolated from the ethanol extract of Kochiae Fructus, and their structures were elucidated by analyzing UV, IR, HR-ESI-MS, 1D, and 2D NMR spectroscopic data. Among them, compounds 6, 8, and 11−17 were isolated for the first time from this plant. The screening results of the glucose uptake experiment indicated that compound 13 had a potent effect on glucose uptake in 3T3-L1 adipocytes at 20 μM. Meanwhile, compounds 3, 9 and 13 exhibited significant inhibitory activities against α-glucosidase, with IC50 values of 23.50 ± 3.37, 4.29 ± 0.52, and 16.99 ± 2.70 µM, respectively, and their α-glucosidase inhibitory activities were reported for the first time. According to the enzyme kinetics using Lineweaver–Burk and Dixon plots, we found that compounds 3, 9 and 13 were α-glucosidase mixed-type inhibitors with Ki values of 56.86 ± 1.23, 48.88 ± 0.07 and 13.63 ± 0.42 μM, respectively. In silico molecular docking analysis showed that compounds 3 and 13 possessed superior binding capacities with α-glucosidase (3A4A AutoDock score: −4.99 and −4.63 kcal/mol). Whereas compound 9 showed +2.74 kcal/mol, which indicated compound 9 exerted the effect of inhibiting α-glucosidase activity by preferentially binding to the enzyme−substrate complex. As a result, compounds 3, 9 and 13 could have therapeutic potentials for type 2 diabetes mellitus, due to their potent hypoglycemic activities.


Introduction
Diabetes mellitus is a chronic metabolic illness that has become a global public health problem [1]. According to the International Diabetes Federation (IDF), approximately 536.6 million adults between the ages of 20 and 79 years had diabetes mellitus in 2021 and this number is expected to rise to 783.2 million by 2045 [2]. Type 2 diabetes mellitus (T2DM) accounts for over 90% of all patients with diabetes and is characterized mainly by insulin resistance, reduction of insulin secretion, and hyperglycemia [3]. It is well established that decreased peripheral glucose uptake, combined with augmented endogenous glucose production, are characteristic features of insulin resistance [4]. Glucosidase enzymes catalyze hydrolysis of starch into simple sugars. In humans, these enzymes aid digestion of dietary carbohydrates and starches to produce glucose for intestinal absorption, which in turn, leads to an increase in blood glucose levels [5]. From these perspectives, enhancing the glucose uptake of organs or tissues [6], as well as inhibiting the activity of α-glucosidase [7], are major strategies for T2DM patients to maintain proper blood glucose levels.

Glucose Uptake and Cell Viability
Using 3T3-L1 adipose model cells, the insulin-induced glucose uptake-enhancing assay was performed to determine glucose consumption after treatment with the compounds. The activity represents the compounds' potential to reduce insulin resistance in body tissues, such as adipocytes, resulting in hypoglycemic effects [43]. To examine the effects of the isolated compounds from KF with glucose uptake in 3T3-L1 adipocytes, 14 compounds (purity ≥ 95%; HPLC) were screened for their abilities to enhance glucose uptake upon the induction of insulin against fully differentiated 3T3-L1 cells. As shown

Glucose Uptake and Cell Viability
Using 3T3-L1 adipose model cells, the insulin-induced glucose uptake-enhancing assay was performed to determine glucose consumption after treatment with the compounds. The activity represents the compounds' potential to reduce insulin resistance in body tissues, such as adipocytes, resulting in hypoglycemic effects [43]. To examine the effects of the isolated compounds from KF with glucose uptake in 3T3-L1 adipocytes, 14 compounds (purity ≥ 95%; HPLC) were screened for their abilities to enhance glucose uptake upon the induction of insulin against fully differentiated 3T3-L1 cells. As shown in Figure 5a, the insulin group could significantly promote the glucose uptake rates of 3T3-L1 adipocytes compared to the control group with a significant difference (p < 0.001). Simultaneously, compound 13 had a strong effect on glucose uptake in 3T3-L1 adipocytes at 20 µM (p < 0.001). Additionally, the results of cell viability showed that compared to the control group, the isolated compounds had no cytotoxicity (Figure 5b). In conclusion, compound 13 had a significant capability to promote glucose uptake in 3T3-L1 adipocytes, and without inhibitory effects on cell viability.
3T3-L1 adipocytes compared to the control group with a significant difference (p < 0 Simultaneously, compound 13 had a strong effect on glucose uptake in 3T3-L1 a cytes at 20 μM (p < 0.001). Additionally, the results of cell viability showed that comp to the control group, the isolated compounds had no cytotoxicity (Figure 5b). In co sion, compound 13 had a significant capability to promote glucose uptake in 3T3-L ipocytes, and without inhibitory effects on cell viability.

α-Glucosidase Inhibition Activity
α-Glucosidase inhibitors exert hypoglycemic effects by slowing the digestio carbohydrates and delaying glucose absorption [44]. Identification of pote α-glucosidase inhibitors were done by in vitro screening of 14 pentacyclic triterp (purity ≥ 95%; HPLC) using an α-glucosidase inhibition experiment. The result shown in Table 3. Compounds 1-9, 11-14, and 17 exhibited varying degree α-glucosidase inhibitory activity, with inhibitory rates between 13.71 ± 0.54 and 74 1.02%. Further tests of α-glucosidase inhibitory activities on compounds 3, 9, and 13 carried out, and the results are shown in Figure 6. Compounds 3, 9, and 13 showe most potent α-glucosidase inhibition activities (p < 0.05) with an IC50 value of 23.50 ± 4.29 ± 0.52, and 16.99 ± 2.70 µM, respectively. This was the first report o α-glucosidase inhibitory activities of compounds 3, 9, and 13. Our study showed compounds 3 and 13 have two hydroxyl groups over the ring of the pentacyclic t penes, which indicated the hydroxyl group in the ring of the pentacyclic triterpenes be an effective functional group as potent α-glucosidase inhibitors. Previous studies reported that the hydroxyl group of pentacyclic triterpenes has been found to con variety of biological properties, such as anti-tumor, anti-inflammatory, antimicrobia hypoglycemic activities [45].

α-Glucosidase Inhibition Activity
α-Glucosidase inhibitors exert hypoglycemic effects by slowing the digestion of carbohydrates and delaying glucose absorption [44]. Identification of potential α-glucosidase inhibitors were done by in vitro screening of 14 pentacyclic triterpenes (purity ≥ 95%; HPLC) using an α-glucosidase inhibition experiment. The results are shown in Table 3. Compounds 1-9, 11-14, and 17 exhibited varying degrees of α-glucosidase inhibitory activity, with inhibitory rates between 13.71 ± 0.54 and 74.41 ± 1.02%. Further tests of α-glucosidase inhibitory activities on compounds 3, 9, and 13 were carried out, and the results are shown in Figure 6. Compounds 3, 9, and 13 showed the most potent αglucosidase inhibition activities (p < 0.05) with an IC 50 value of 23.50 ± 3.37, 4.29 ± 0.52, and 16.99 ± 2.70 µM, respectively. This was the first report of the α-glucosidase inhibitory activities of compounds 3, 9, and 13. Our study showed that compounds 3 and 13 have two hydroxyl groups over the ring of the pentacyclic triterpenes, which indicated the hydroxyl group in the ring of the pentacyclic triterpenes may be an effective functional group as potent α-glucosidase inhibitors. Previous studies also reported that the hydroxyl group of pentacyclic triterpenes has been found to confer a variety of biological properties, such as anti-tumor, anti-inflammatory, antimicrobial and hypoglycemic activities [45]. Data were expressed as the mean value ± SD (n = 3); means followed by the different letters ( a−j ) are significantly different (p < 0.05); A , percent inhibition at a concentration of 0.012 μM; B , percent inhibition at a concentration of 50 μM.

Enzyme Kinetic Equation
It is well accepted that enzyme kinetics can provided some useful information for predicting the interactions between the ligands and enzymes. To clarify how and where triterpenes bind to α-glucosidase, we first measured the enzyme kinetics of compounds 3, 9 and 13, by using methods similar to those described in the literature [46]. As shown in Figure

Enzyme Kinetic Equation
It is well accepted that enzyme kinetics can provided some useful information for predicting the interactions between the ligands and enzymes. To clarify how and where triterpenes bind to α-glucosidase, we first measured the enzyme kinetics of compounds 3, 9 and 13, by using methods similar to those described in the literature [46]. As shown in Figure 7, the concentrations of 1/[pNPG] are displayed on the X-axis, and 1/v values obtained from the Lineweaver-Burk plot are shown along the Y-axis. The plots of compound 3 intersected in the second quadrant, meaning that the Vmax values decreased and the Km values increased with the increased concentration of inhibitors (Figure 7a). The plots of compounds 9 and 13 intersected in the third quadrant, meaning that both the Km and Vmax values decreased with the increased concentration of inhibitors (Figure 7b,c). The results indicated that compounds 3, 9 and 13 caused a mixed-type inhibition, which meant they could bind to both the free enzyme and the enzyme-substrate complex [47].
We also examined Dixon plots of how compounds 3, 9 and 13 affect α-glucosidase. As shown in Figure 8, these plots further confirmed that compounds 3, 9 and 13 are mixed-type α-glucosidase inhibitors. The K i values of compounds 3, 9 and 13 were 56.86 ± 1.23, 48.88 ± 0.07 and 13.63 ± 0.42 µM, respectively, while the K i ' values of these compounds were 47.89 ± 1.37, 19.52 ± 0.26 and 8.82 ± 0.06 µM, respectively. K i is the equilibrium constant for the inhibitor binding to α-glucosidase, and K i ' is the equilibrium constant for the inhibitor binding to the α-glucosidase−pNPG complex [48]. The results showed that the K i ' values were smaller than the K i values, which indicated that the inhibitor−enzyme−substrate complex binding affinity exceeds the binding affinity of the inhibitor−enzyme. The binding sites and mechanism underlying inhibition have yet to be determined. However, the results of compounds 3, 9 and 13 bound to either α-glucosidase, or the α-glucosidase−pNPG complex, further confirmed that compounds 3, 9 and 13 are mixed−competitive inhibition against α-glucosidase. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW Km and Vmax values decreased with the increased concentration of inhibitors 7b,c). The results indicated that compounds 3, 9 and 13 caused a mixed-type inh which meant they could bind to both the free enzyme and the enzyme-substrate co [47]. We also examined Dixon plots of how compounds 3, 9 and 13 affect α-gluco As shown in Figure 8, these plots further confirmed that compounds 3, 9 and mixed-type α-glucosidase inhibitors. The Ki values of compounds 3, 9 and 13 wer ± 1.23, 48.88 ± 0.07 and 13.63 ± 0.42 μM, respectively, while the Ki' values of thes pounds were 47.89 ± 1.37, 19.52 ± 0.26 and 8.82 ± 0.06 μM, respectively. Ki is the e rium constant for the inhibitor binding to α-glucosidase, and Ki' is the equilibriu stant for the inhibitor binding to the α-glucosidase−pNPG complex [48]. The showed that the Ki' values were smaller than the Ki values, which indicated that hibitor−enzyme−substrate complex binding affinity exceeds the binding affinity inhibitor−enzyme. The binding sites and mechanism underlying inhibition have be determined. However, the results of compounds 3, 9 and 13 bound to α-glucosidase, or the α-glucosidase−pNPG complex, further confirmed that comp 3, 9 and 13 are mixed−competitive inhibition against α-glucosidase.

Molecular Docking
Molecular docking is a key tool in structural molecular biology and computer-assisted drug design. The goal of ligand-protein docking is to predict the predominant binding modes of a ligand with a protein of known 3D structure [49]. The calculated binding energies of 2β,3β-dihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranoside (3), and 22β-hydroxy-oleanolic acid (13) with α-glucosidase, were found to be −4.99 and −4.63 kcal/mol, respectively. But the binding energy of compound 9 with α-glucosidase was found to be +2.74 kcal/mol, which indicated poor binding (Figure 9). This result further suggested that compound 9 exerted the effect of inhibiting α-glucosidase activity by preferentially binding to the enzyme-substrate complex. This coincided with the results of the enzyme kinetic analysis, in which the K i and K i ' values of compound 9 were 48.88 ± 0.07 and 19.52 ± 0.26 µM, respectively. Interestingly, significant H-bonding interactions with the hydroxyl groups of compounds 3 and 13 were found in all these binding sites. For compound 3, there are four residues (Asn 247, Thr 285, Ser 282 and Asp 242) which formed six hydrogen bonds with the compound. Among these, the hydrogen of the hydroxyl groups at the C-2 and C-3 position on the ring A of compound 3 formed four hydrogen-bonding interactions with Asn 247, Thr 285, Ser 282 residues of the enzyme ( Figure 10). For compound 13, only one hydrogen bond was formed between the compound and the residues of α-glucosidase. It was established between the hydrogen of the hydroxyl group at the C-3 position on the ring A of compound 13 and Arg 359, with a distance of 2.1 Å (Figure 10). This accounts well for the previous observation that hydroxyl groups were essential to improve the inhibitory activity of the compound.

Molecular Docking
Molecular docking is a key tool in structural molecular biology and com er-assisted drug design. The goal of ligand-protein docking is to predict the predom binding modes of a ligand with a protein of known 3D structure [49]. The calcu binding energies of 2β,3β-dihydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyran (3), and 22β-hydroxy-oleanolic acid (13) with α-glucosidase, were found to be −4.9 −4.63 kcal/mol, respectively. But the binding energy of compound 9 with α-glucos was found to be +2.74 kcal/mol, which indicated poor binding (Figure 9). This further suggested that compound 9 exerted the effect of inhibiting α-glucosidase ac by preferentially binding to the enzyme-substrate complex. This coincided with th sults of the enzyme kinetic analysis, in which the Ki and Ki' values of compound 9 48.88 ± 0.07 and 19.52 ± 0.26 μM, respectively. Interestingly, significant H-bonding the hydroxyl groups at the C-2 and C-3 position on the ring A of compound 3 formed four hydrogen-bonding interactions with Asn 247, Thr 285, Ser 282 residues of the enzyme ( Figure 10). For compound 13, only one hydrogen bond was formed between the compound and the residues of α-glucosidase. It was established between the hydrogen of the hydroxyl group at the C-3 position on the ring A of compound 13 and Arg 359, with a distance of 2.1 Å (Figure 10). This accounts well for the previous observation that hydroxyl groups were essential to improve the inhibitory activity of the compound.

Chemicals, Reagents and Cell
1 H and 13 C and 2D NMR spectra were obtained on a Bruker-Avance Ⅲ-500 MHz (Bruker Corporation, Madison, WI, USA) spectrometer with chemical shifts recorded in δ (ppm), using tetramethylsilane (TMS) as the internal standard, while the coupling constants (J) were given in hertz. Mass spectra were obtained on an MS Waters AutoSpec Premier P776 mass spectrometer (ESI-MS) and a UPLC-IT-TOF-MS (HR-ESI-MS), re- the hydroxyl groups at the C-2 and C-3 position on the ring A of compound 3 formed four hydrogen-bonding interactions with Asn 247, Thr 285, Ser 282 residues of the enzyme ( Figure 10). For compound 13, only one hydrogen bond was formed between the compound and the residues of α-glucosidase. It was established between the hydrogen of the hydroxyl group at the C-3 position on the ring A of compound 13 and Arg 359, with a distance of 2.1 Å (Figure 10). This accounts well for the previous observation that hydroxyl groups were essential to improve the inhibitory activity of the compound.

Chemicals, Reagents and Cell
1 H and 13 C and 2D NMR spectra were obtained on a Bruker-Avance Ⅲ-500 MHz (Bruker Corporation, Madison, WI, USA) spectrometer with chemical shifts recorded in δ (ppm), using tetramethylsilane (TMS) as the internal standard, while the coupling constants (J) were given in hertz. Mass spectra were obtained on an MS Waters AutoSpec Premier P776 mass spectrometer (ESI-MS) and a UPLC-IT-TOF-MS (HR-ESI-MS), re-

Glucose Uptake and Cell Viability Assays
The mature adipocytes were inoculated in a 96-well plate at 5 × 10 4 cells per well for 24 h. The 3T3-L1 adipocytes were divided into model groups (blank control), insulin group (250 ng/mL, positive control), and sample group (20 µM). After 24 h of administration, 10 µL medium was used to measure the glucose content. The absorbance value was detected by a microplate reader at 505 nm. The glucose uptake rate was calculated as follows: where A 1 is the absorbance of the mixed-glucose, DMEM-supplemented control, and A 2 is the absorbance of the blank, insulin, or sample group.

Glucose Uptake and Cell Viability Assays
The mature adipocytes were inoculated in a 96-well plate at 5 × 104 cells per we 24 h. The 3T3-L1 adipocytes were divided into model groups (blank control), in group (250 ng/mL, positive control), and sample group (20 μM). After 24 h of ad istration, 10 μL medium was used to measure the glucose content. The absorbance v was detected by a microplate reader at 505 nm. The glucose uptake rate was calculat follows: Glucose uptake rate (%) = [1 − A2/A1] × 100 where A1 is the absorbance of the mixed-glucose, DMEM-supplemented control, an is the absorbance of the blank, insulin, or sample group.
Cell viability was detected by using a CellTiter 96 ® AQueous One Solution Cell liferation Assay (Promega Corporation, Madison, WI, USA) according to the man turer's instructions after the glucose uptake experiment [50]. CellTiter 96 ® AQueous Solution Cell Proliferation Assay reagent (20 µL/well) was added to the plate and bated at 37 °C for 180 min before absorbance was measured at 490 nm, and the rel cell viability was calculated as follows: Relative cell viability (%) = A1/A2 × 100 where A1 is the absorbance of the blank control, and A2 is the absorbance of the sa group.
Cell viability was detected by using a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions after the glucose uptake experiment [50]. CellTiter 96 ® AQueous One Solution Cell Proliferation Assay reagent (20 µL/well) was added to the plate and incubated at 37 • C for 180 min before absorbance was measured at 490 nm, and the relative cell viability was calculated as follows: Relative cell viability (%) = A 1 /A 2 × 100 (2) where A 1 is the absorbance of the blank control, and A 2 is the absorbance of the sample group.

In Vitro α-Glucosidase Inhibition Assay
The α-glucosidase inhibition assay was carried out on the basis of the method reported by Zhang et al., with minor modifications [51]. Briefly, α-glucosidase (0.1 U/mL), pNPG (5 mM) and Na 2 CO 3 (0.5 M) were prepared in PBS (0.1 M, pH 6.8), and the samples were diluted to different concentrations (0.0625, 0.125, 0.25, 0.5, 1, and 2 mM) using PBS. In a 96-well microplate, a mixture of 80 µL PBS, 10 µL sample, and 50 µL α-glucosidase solution were added and incubated for 15 min at 37 • C, 10 µL PBS used as a blank control. To initiate the reaction, pNPG (40 µL) was added to the reaction mixture and incubated at 37 • C for 30 min. The reaction was terminated by adding 20 µL of Na 2 CO 3 , after which the absorbance was determined at 405 nm by a microplate reader (SpectraMax190, Micro-g Biotech, Guangzhou, China). Acarbose was used as a positive control in this α-glucosidase inhibition assay. IC 50 values were defined as the concentration of the compound required to inhibit 50% of α-glucosidase activity under assay conditions. The α-glucosidase inhibition activity was calculated as follows: Inhibition rate (%) = (A 1 − A 2 )/A 1 × 100 where A 1 is the OD value of the blank control, A 2 is the OD value of the tested samples, and the analysis was performed in triplicates.

Kinetics Involved in the Inhibition of α-Glucosidase
The kinetic analysis of compounds 3, 9, and 13 were measured using the reaction conditions in Section 3.6. Typically, three different concentrations of each compound around the IC 50 values were chosen. Under each concentration, α-glucosidase activity was assayed by varying the concentration of pNPG as a substrate [46]. The inhibition types of active compounds were determined by Lineweaver-Burk plots [the inverse of velocity (1/v) against the inverse of the substrate concentration (1/[pNPG])] with substrate concentrations of 1.25, 2.5, 5, 10, 20 µM. K i and K i ' values were determined from 1/v versus [I] (Dixon plot) and S/v versus [I] plots, respectively.

Molecular Docking
The molecular docking approach can be used to model the interaction between a small molecule and a protein at the atomic level [52]. The structure of α-glucosidase (PDB ID: 3A4A) was obtained from the Online Protein Data Bank [53], and the 3D structures of the ligands were generated by Chem3D Pro (version: 14.0). Complexed ligands and water molecules in the crystal structure of α-glucosidase were virtually removed by PyMOL Win application (PyMOL, version: 2.4.0). Gasteiger charges and essential hydrogen atoms were added by using the AutoDock tools (ADT, version: 1.5.6). The cubic grid box dimensions of α-glucosidase were defined as x = 98, y = 126, and z = 102 Å with spacing of 0.692 Å. Finally, the PyMOL molecular graphics system (version 2.4.0) was used to visualize ligandenzyme interactions.

Statistical Analysis
IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA), was used to analyze all of the data. The experiments were carried out in triplicates and the results were expressed as an average of the three measurements ± SD. One-way analysis of variance (ANOVA) was used to compare the means of different analysis investigations. Differences were considered significant when * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
Seventeen compounds, including three previously undescribed and fourteen known triterpenes, were isolated from the ethanol extract of KF. We detected their hypoglycemic activities via assays for α-glucosidase inhibition and glucose uptake of 3T3-L1 adipocytes. Next, we performed enzyme kinetics and molecular docking investigations to analyze the possible mechanisms against enzymes. The results of the glucose uptake experiment showed compound 13 had a significant promotion on glucose uptake rate of 3T3-L1 adipocytes (p < 0.001). Simultaneously, enzyme-inhibition results suggested that compounds 3, 9, and 13 possessed potent inhibitory effects on α-glucosidase, and their enzymatic kinetics on α-glucosidase showed that they are mixed-type inhibitors. The hydroxyl group in the ring of the pentacyclic triterpene played a key role in maintaining α-glucosidase inhibitory activity according to the docking simulation. In summary, this study enriched the chemical composition diversity of KF and provided effective evidence for its use in hypoglycemic herbal medicine.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
All data presented in this study are available in the article.