In Vitro and In Silico Analysis of PTP1B Inhibitors from Cleistocalyx operculatus Leaves and Their Effect on Glucose Uptake

As part of our ongoing research on new anti-diabetic compounds from ethnopharmacologically consumed plants, two previously undescribed lupane-type triterpenoids (1 and 2) with dicarboxylic groups, an undescribed nor-taraxastane-type triterpenoid (3), and 14 known compounds (4–17) were isolated from the leaves of Cleistocalyx operculatus. Extensive spectroscopic analysis (IR, HRESIMS, 1D, and 2D NMR) was used for structure elucidation, while the known compounds were compared to reference data reported in the scientific literature. All the isolates (1–17) were evaluated for their inhibitory effects on the protein tyrosine phosphatase 1B (PTP1B) enzyme. Compounds 6, 9, and 17 showed strong PTP1B inhibitory activities. The mechanism of PTP1B inhibition was studied through enzyme kinetic experiments. A non-competitive mechanism of inhibition was determined using Lineweaver–Burk plots for compounds 6, 9, and 17. Additionally, Dixon plots were employed to determine the inhibition constant. Further insights were gained through a structure–activity relationship study and molecular docking analysis of isolated compounds with the PTP1B crystal structure. Moreover, all isolates (1–17) were tested for their stimulatory effects on the uptake of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) in differentiated 3T3-L1 adipocyte cells. Compounds 6, 13, and 17 exhibited strong glucose absorption stimulation activity in a dose-dependent manner.


Introduction
The prevalence of diabetes has been steadily increasing globally in recent decades, with an estimated 10.5% of individuals aged between 20 and 79 diagnosed with the disease in 2021 [1].Within this age range, diabetes is a risk factor in one out of every nine deaths resulting from complications associated with the condition, underscoring its significance as a major public health concern worldwide [2,3].Despite the development of numerous non-insulin glucose-lowering agents for managing diabetes, controlling target blood glucose levels remains challenging and often results in undesirable side effects [4,5].Hence, there is an ongoing need to investigate new substances to prevent or treat diabetes more effectively.Insulin resistance is a central factor in the pathogenesis of type 2 diabetes [6].In the manifestation of insulin resistance, protein tyrosine phosphatase 1B (PTP1B) plays a significant role as a negative regulator of insulin signaling [7][8][9][10][11].Thus, PTP1B has been identified as a promising target for developing improved glucose-regulating agents [12].
Medicinal plants have been used empirically and reported to be helpful in diabetes treatment [13,14].The mechanisms of action attributed to reported anti-diabetic plants include insulin mimetics, increased insulin secretion, β-cells regeneration in the pancreas, reduced glucose absorption, reduced glucosidase and amylase activity, and radical scavenging activity [13,15].Additionally, it has been observed that some plant extracts and their constituents exhibit PTP1B inhibitory action [16].However, despite many research efforts, no approved medication currently targets PTP1B inhibition.Cleistocalyx operculatus (Roxb.Merry.Et Perry) is an evergreen plant that has been historically utilized in Vietnam and China as traditional herbal tea and medicine to treat various ailments such as colds, fever, dysentery, vomiting, indigestion, gastrointestinal disorders, and inflammation [17][18][19][20].Interestingly, an aqueous extract from flower buds of C. operculatus was reported to decrease glucose levels in individuals who were administered the extract for 12 weeks [21].Chalcone-meroterpene derivatives isolated from the buds of C. operculatus have shown PTP1B inhibition activity [22].Other chemical constituents identified in the plant include flavanones, flavones, and oleanane-and ursane-type triterpenoids [23][24][25].

Plant Material
The leaves of Cleistocalyx operculatus were purchased from a market in My Duc district, Hanoi, Vietnam, in February 2019.The botanical identification of the specimen was performed by Prof. Won Keun Oh.A voucher specimen (SNU2019-02) was deposited at the Laboratory of Pharmacognosy, College of Pharmacy, Seoul National University, Seoul, Republic of Korea.

In Vitro PTP1B Inhibition Assay
PTP1B enzyme inhibition activity was assessed following a previously described protocol [26].Briefly, 50 µL of 4 mM p-NPP was added to a buffer solution containing 1 mM dithiothreitol (DTT), 0.1 M NaCl, 1 mM EDTA, 50 mM citrate (pH 6.0), and 16 nM PTP1B (Enzo Life Sciences Inc., Farmingdale, NY, USA), along with either the test compounds or dimethyl sulfoxide (DMSO), bringing the final volume to 100 µL in a 96-well half-volume plate.It was followed by incubation at 37 • C for 30 min.After that, 10 µL of 10 M NaOH was added to quench the reaction.The enzymatic reaction product was measured by measuring the absorbance in a UV plate reader () at 405 nm.Ursolic acid was utilized as a positive control.The non-enzymatic reaction of the substrate was considered and accounted for by comparing data to a control experiment without the PTP1B enzyme.IC 50 values were calculated using non-linear regression in GraphPad Prism 10 (GraphPad Software, Inc., San Diego, CA, USA), based on triplicate experiments.

Kinetic Analysis with PTP1B
Reaction velocity was measured at different concentrations of tested compounds for kinetic analysis.The tested compounds or DMSO were preincubated with the enzyme and buffer solution at 37 • C for 2 min, followed by adding 50 µL of substrate (p-NPP) at different concentrations.The initial reaction velocities were monitored at 37 • C at 405 nm for 15 min.The kinetic reaction slopes were calculated based on the absorbance increments observed between 2 and 10 min (within the linear range).GraphPad Prism 10 (GraphPad Software, Inc., San Diego, CA, USA) was used to graph the double reciprocal Lineweaver-Burk and Dixon plots.

Measurement of Glucose Uptake Using the 2-NBDG Probe
In vitro experiments measured glucose uptake in 3T3-L1 adipocytes using 2-NBDG, a fluorescent glucose derivative (Invitrogen, Eugene, OR, USA).Cells were cultured on 96-well plates in a glucose-free medium supplemented with 10% FBS.After incubation, cells were treated with either insulin (as a positive control) or the test compounds in the presence or absence of 2-NBDG and were further incubated for an additional hour.Cells were washed with cold phosphate-buffered saline (PBS).The 2-NBDG fluorescence was quantified by measuring the signal intensity at excitation/emission wavelengths of 450/535 nm using a SpectraMax Gemini XPS fluorescence microplate reader (Molecular Devices, San Jose, CA, USA).For cellular transport studies involving 2-NBDG, 3T3-L1 adipocytes were grown on sterilized glass coverslips in a glucose-free medium containing 10% FBS for 24 h.After treatment with 2-NBDG, cells were washed with cold PBS.Fluorescent images were obtained with an Olympus IX 70 fluorescence microscope (Olympus Corporation, Tokyo, Japan) to quantify the cellular transport of 2-NBDG.

Molecular Docking Studies on the PTP1B Enzyme
The chemical structures of compounds 1-17 intended for docking simulations were minimized using Chem3D (Perkin Elmer, Shelton, CT, USA) and saved in .molformat.These files were then imported into Discovery Studio (Dassault Systèmes Biovia Corp., Vélizy-Villacoublay, France), where various conformers were generated, and ionization states were produced using the 'Prepare Ligands' tool.Energy minimization was performed by applying CHARMm force field and the Momany-Rone method.The structural data of the PTP1B protein were acquired from the RCSB Protein Data Bank, referencing PDB:1T49 [27].For the preparation of the protein structure, parameters were set to CHARMm minimization, with protonation adjusted to pH 7.4, an ionic strength of 0.145, and the removal of water molecules.The docking procedure was conducted employing the Libdock protocol [28].The coordinates set for identifying the sphere of protein-ligand interactions within the allosteric binding site of PTP1B were as follows: 53.8545, 30.4053, 24.5127, and 8.9.Default parameters were selected for docking preferences, setting the quality to high, choosing the FAST method for conformation, and applying the Steepest Descent for the Minimization Algorithm.Finally, the Discovery Studio Visualizer (Dassault Systèmes Biovia Corp., Vélizy-Villacoublay, France) was used to display the binding poses, protein surfaces, and interactions between proteins and ligands.

Statistical Analysis
Data were processed through variance analysis (ANOVA) to determine the significance of differences between groups, followed by Tukey's or Duncan's post hoc test.A p-value < 0.05 was considered indicative of a significant difference, with levels of significance denoted as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

PTP1B Inhibitory Activity of Compounds 1−17 and Enzyme Kinetics
PTP1B has been established as an attractive drug target for treating diabetes [39][40][41].To explore possible therapeutic potentials, we measured the inhibitory effects of compounds 1-17 on the PTP1B enzyme, with ursolic acid as a positive control (Figure S22).Firstly, we screened all isolated compounds at a concentration of 20 µM.Most isolates exhibited inhibitory activity against PTP1B except for cleistocalyxic acid L (1), cleistocalyxolide C (3), ulmoidol (7), and (2a,3b,12a)-trihydorxy-olean-28-oic acid 28,13-lactone (8).From this result, it can be inferred that the presence of a lactone ring in 3, 7, and 8 is associated with reduced PTP1B inhibitory activity.Following the initial screening, the half-maximal response of PTP1B activity (IC 50 ) was determined for the compounds that showed less than 50% enzyme activity at a concentration of 20 µM.To determine the IC 50 values, we employed six different concentrations for each compound to establish their inhibitory potency profiles.The results revealed that betulinic acid (5), oleanolic acid (12), maslinic acid (13), and 3-O-(Z)-p-coumaroyl maslinic acid (17) exhibited significant inhibitory activity on the PTP1B enzyme, similar to the positive control (Table 2 and Figures S23-S26).The PTP1B inhibitory effects of betulinic acid, oleanolic acid, and maslinic acid have been previously reported, and our results support these previous reports.
We selected one compound each from different triterpenoid backbones, including lupane, ursane, and oleanane types, for further study.The enzymatic inhibition mechanism of the selected compounds 6, 9,13, and 17 was deduced from experimental kinetic data, where different concentrations of the p-NPP substrate and inhibitors were tested to measure the velocities of the PTP1B enzymatic reaction.The inverse of the velocity and the inverse of the substrate concentration values were used to create Lineweaver-Burk plots.These plots showed straight lines intersecting on the 1/[S] axis for compounds 6, 9, 13, and 17 (Figure 3), indicating that these compounds act as non-competitive inhibitor of PTP1B.As a lupane-type triterpenoid, compound 6 shares the same skeletal structure with compounds 1, 2, 4, and 5. Compound 9 shows structural similarities with compounds 10 and 11, while compounds 12-17 are oleanane-type triterpenoids.It is reasonable to assume that all the isolated compounds possess a non-competitive mechanism of action in inhibiting PTP1B since compounds 6, 9, 13, and 17, which are representative compounds of each type, showed non-competitive inhibition of PTP1B.We further examined Dixon plots, which depict the plot of 1/enzyme velocity (1/V) against inhibitor concentration (I), to determine the dissociation or inhibition constant (K i ) for the enzyme-inhibitor complex.The value at which the x-axis intersects corresponds to-K i .The K i values for compounds 6, 9, 13, and 17 were calculated to be 2.8 ± 0.4, 7.6 ± 1.2, 3.2 ± 0.6, and 2.7 ± 0.4 µM, respectively (Figure 3, Table 2).The K i value serves as an important indicator of the binding affinity between an enzyme and an inhibitor.For non-competitive inhibitors, the IC 50 is expected to equal the K i .Our experimental data show that the values obtained for both parameters are consistent (Table 2).

Structure Activity Relationship (SAR) Analysis and Molecular Docking Experiments
The isolated compounds predominantly feature a pentacyclic triterpenoid ring, enabling us to discern the impact of various functional groups within this structural framework on PTP1B inhibition.Compounds 3, 7, and 8 exhibit reduced inhibitory effects, likely due to the presence of a lactone ring connecting C-13 and C-18.Similarly, hydroxylation of methyl groups at Me-24 and Me-30 leads to diminished activity among lupane-type triterpenes, including compounds 1, 2, and 4-6.Adding functional groups to the ursolic acid skeleton, such as the C-3 and C-19 hydroxyl groups in compounds 9 and 10, results in a slight reduction in PTP1B inhibition.Additionally, a decrease in activity is observed with methyl modifications at C-24 and C-25 in compounds 10 and 11, leading to a more noticeable decline in bioactivity compared to compound 9 and ursolic acid.The inhibition efficacy of oleanolic acid ( 12) is slightly improved by incorporating hydroxyl groups at position C-2.Furthermore, substituting the carboxylic acid at C-28 with a hydroxyl group diminishes its activity.Adding a p-coumaroyl moiety to maslinic acid (13) results in a slight decrease in activity for compounds 16 and 17.Molecular docking simulations were employed to better understand the chemical interactions between the isolated compounds and the PTP1B protein.The selection of the 1T49 crystal structure for molecular docking studies was based on prior published research and the presence of a crystallized ligand at the allosteric site [27,42,43].The docking results yielded various binding poses for the isolated molecules (Table 3).A detailed evaluation was performed, and the pose with the most favorable score was chosen for further examination.Most molecules exhibited binding poses that fit well into the hydrophobic pocket of the allosteric site (Figure S27).In the case of lupane derivatives, key interactions were observed between the olefinic carbons and the amino acids leucine-192 and phenylalanine-196, resulting in a π-alkyl interaction.Hydroxylation at C-30 may hinder this interaction, as seen in compound 1.Alkyl interactions were also noted between Me-25 and the backbone of lysine-197.Moreover, conventional hydrogen bond interactions were confirmed between the carboxylic acid and alanine-193 and lysine-197.Substituting it with a methyl ester carboxylic acid appears to eliminate this interaction.The additional hydroxyl group at position Me-24 in ursane-type triterpenoids may facilitate other interactions, such as conventional hydrogen bonds, as evidenced by the interaction between this hydroxyl group and glutamate-276 (Figure 4).Additional hydrogen bond interactions between the carboxylic acid and alanine-193 and lysine-197 were observed for ursane-and oleananetype triterpenoids.Similarly, compound 13 showed hydrophobic interactions with Phe280 and Ala 189.Also, there were hydrogen bond interactions between the carboxylic acid group and Lys197.The inclusion of a coumaroyl substituent at position C-3, as seen in compound 16 and 17, resulted in binding poses with additional π-π interactions between the aromatic ring in the coumaroyl moiety and phenylalanine-280.These poses showed better docking scores than compounds 13.However, this did not translate into better performance in the observed experimental enzymatic inhibitory activity.In conclusion, preserving an unmodified hydrophobic core is crucial for maintaining inhibitory activity in PTP1B.The incorporation of polar groups at the core, particularly in the C and D rings, results in a reduction in binding affinity.A carboxylic acid moiety at C-28 is also pivotal in facilitating hydrogen bonding with polar protein residues.Substituents at C-3, as demonstrated by compounds 16 and 17, did not significantly affect enzymatic activity and can contribute to enhanced binding affinity.These insights into the structural determinants of PTP1B inhibition provide valuable information for developing future derivatives based on isolated triterpene scaffolds.

Effect of Stimulating Glucose Uptake in Adipocytes
To determine if the PTP1B inhibition activity of compounds 1-17 affects the glucose uptake process, we also further evaluated their effects in vitro using 2-NBDG in 3T3-L1

Effect of Stimulating Glucose Uptake in Adipocytes
To determine if the PTP1B inhibition activity of compounds 1-17 affects the glucose uptake process, we also further evaluated their effects in vitro using 2-NBDG in 3T3-L1 adipocyte cells.2-NBDG is a known fluorescent-tagged glucose probe used for identifying insulin-mimetic compounds [44,45].The 3T3-L1 adipocytes, insulin-sensitive cells that are fully differentiated, were utilized for this experiment.To determine the transportation efficacy of 2-NBDG into the cells, we assessed the fluorescent signal in the differentiated adipocytes after treating them with each isolate at a concentration of 20 µM using fluorescence microscopy, with insulin serving as a positive control (Figure 5).As a result, compounds 6, 12, 13, 16, and 17, which also exhibited PTP1B inhibitory activities, showed glucose uptake stimulatory effects.Among them, compounds 6, 13 and 17 displayed particularly potent glucose uptake stimulatory effects, prompting further investigation for these compounds at various concentrations (5, 10, and 20 µM).The activities of compounds 6, 13, and 17 on glucose uptake were found to be dose dependent (Figure 5).

Effect of Stimulating Glucose Uptake in Adipocytes
To determine if the PTP1B inhibition activity of compounds 1-17 affects the glucose uptake process, we also further evaluated their effects in vitro using 2-NBDG in 3T3-L1 adipocyte cells.2-NBDG is a known fluorescent-tagged glucose probe used for identifying insulin-mimetic compounds [44,45].The 3T3-L1 adipocytes, insulin-sensitive cells that are fully differentiated, were utilized for this experiment.To determine the transportation efficacy of 2-NBDG into the cells, we assessed the fluorescent signal in the differentiated adipocytes after treating them with each isolate at a concentration of 20 µM using fluorescence microscopy, with insulin serving as a positive control (Figure 5).As a result, compounds 6, 12, 13, 16, and 17, which also exhibited PTP1B inhibitory activities, showed glucose uptake stimulatory effects.Among them, compounds 6, 13 and 17 displayed particularly potent glucose uptake stimulatory effects, prompting further investigation for these compounds at various concentrations (5, 10, and 20 µM).The activities of compounds 6, 13, and 17 on glucose uptake were found to be dose dependent (Figure 5).

Discussion
PTP1B is a promising target for diabetes control due to its role in the development of insulin resistance, where it plays a key role in regulating insulin signaling.Triterpenes have been proven to possess anti-diabetic potential and have inhibitory effects on PTP1B [16,46,47].Several studies have highlighted the ability of triterpenoids to improve glucose uptake [44,45].Oleanolic acid (12) has been shown to improve glucose homeostasis and prevent the progression of type 2 diabetes in pre-diabetic male Sprague Dawley rats [44].Furthermore, maslinic acid (13), a triterpenoid isolated in this study, increased glucose uptake, reduced lipid droplet and triglyceride levels, and raised intracellular Ca 2+ concentration, suggesting its potential as a candidate for obesity and diabetes treatment [48].Among the triterpenes in C. operculatus leaves, maslinic acid, corosolic acid, asiatic acid, and arjulonic acid are the most abundant [24].The content of total terpenoids in hot-water infusion from C. operculatus leaves has been reported as 13.12 mg/g of dried leaves [49].Thus, it is natural to assume that C. operculatus leaves may exert anti-diabetic properties, but no prior research has reported it.
In this study, the isolation of 17 triterpenes with various substitution patterns facilitated the elucidation of their structure-activity relationships concerning glucose uptake efficacy and PTP1B inhibition.Interestingly, we found that compounds inhibiting PTP1B also stimulated glucose uptake.A moderate positive correlation was observed between glucose uptake activity and PTP1B inhibition (Spearman correlation, R = 0.51, p = 0.03; see Figure S28).This suggests that changes affecting PTP1B inhibitory activity will likely influence glucose uptake stimulation.Therefore, the stimulatory effect on glucose uptake appears to be mediated through PTP1B.It is well established that the dephosphorylation of insulin receptor substrates (IRS) by PTP1B affects the translocation of glucose transporter type 4 (GLUT4), significantly reducing glucose uptake in adipose and skeletal muscle cells [50][51][52][53][54][55].This known mechanism supports the correlation observed in our experimental results.
Betulinic acid (5) showed slightly better activity than alphitolic acid (6) in inhibiting PTP1B.However, the additional hydroxyl group at C-2 significantly improved glucose uptake activity of 6, likely due to enhanced solubility or transport through cell membranes (Table S1).Similarly, the hydroxyl group at C-2 improved the activity of maslinic acid (13) compared to oleanolic acid (12).Hydroxylation at other positions compromises PTP1B inhibition and glucose uptake stimulation.On the other hand, adding a p-coumaroyl moiety to maslinic acid (13) did not significantly impact either PTP1B inhibitory activity or glucose uptake.Modifications of ursolic acid skeleton reduced PTP1B inhibition for compounds 9-11.Compounds 3, 7, and 8 exhibited low glucose uptake stimulation and PTP1B inhibition activity, likely due to a lactone ring connecting C-13 and C-18.Despite the known bioactivities of betulinic acid (5), oleanolic acid (12), maslinic acid (13), and ursolic acid on PTP1B, we further confirmed their effects on glucose uptake stimulation.In addition, we observed that, in general, compounds retained the same levels of bioactivity.However, alphitolic acid (6), maslinic acid (13), and 3-O-(Z)-p-coumaroyl maslinic acid (17) outperformed the others in the glucose uptake assay in 3T3-L1 adipocytes, possibly due to better solubility and the ability to absorb through cell membranes.
Overall, we anticipate that the findings of this study will contribute significantly to the development of standardized extracts of C. operculatus as a promising option for complementary diabetes treatment.

Conclusions
During our investigation of Cleistocalyx operculatus leaves, we isolated three new triterpenoids (1−3) and 14 known compounds (4−17).All these compounds were tested in PTP1B inhibition assays, and their IC 50 values were determined.The most active compounds were betulinic acid (5), oleanolic acid (12), and maslinic acid (13).Enzyme kinetic experiments revealed that compounds possess a non-competitive inhibition mechanism, which molecular docking models supported.In vitro glucose uptake assays in differentiated 3T3-L1 adipocytes showed that alphitolic acid (6), maslinic acid (13), and 3-O-(Z)-p-coumaroyl maslinic acid (17) exhibited the most potent anti-diabetic effects.In addition, we infer that the glucose uptake stimulation effect by the isolated compounds is mediated through PTP1B, as indicated by the moderate positive correlation.Overall, this work provides experimental evidence supporting the potential of C. operculatus leaf extract as a plant-based alternative for diabetes management.S1.ADMET profile of isolated compounds predicted by QSAR regression models.

Figure 1 .
Figure 1.Chemical structures of isolated compounds 1−17 from the leaves of C. operculatus.

Figure 1 .
Figure 1.Chemical structures of isolated compounds 1−17 from the leaves of C. operculatus.Compound 2 was obtained as a white amorphous powder, with its molecular formula determined as C 31 H 48 O 6 , based on HRESIMS data (m/z 515.3372 [M − H] -, calcd for 515.3373).The IR spectrum revealed absorption bands indicative of hydroxyl (3245 cm -1 ), ester (1718 and 1212 cm -1 ), and carboxyl (2934 and 1680 cm -1 ) groups.The 1 H and 13 C NMR spectra of compound 2 (Table 1) were similar to those of 1, with the only difference being the change in the position of a hydroxyl group.The chemical shifts from δ H 4.73 and 4.56 at C-30 in compound 1 had disappeared, and new chemical shifts at δ H 4.10 and 3.59 at the C-23 in compound 2 appeared.Based on HSQC, two correlations from δ H 4.10 and 3.59 (H-23) to C-23 (δ C 68.5) suggested the presence of a hydroxyl group at C-23.HMBC correlations from H-23 to C-3 (δ C 74.0), C-4 (δ C 43.3), C-5 (δ C 49.6), and C-24 (δ C 13.5) clearly indicated the position of the hydroxyl group at C-23.Additionally, HMBC correlations from H-22 (δ H 2.02 and 1.45)/H-18 (δ H 2.13)/H-16 (δ H 1.88) and the methoxy group (δ H 3.77) to C-28 (δ C 177.3) determined the position of methyl ester carboxylic acid.The HMBC correlations from H-13 (δ H 2.71) and H-15 (δ H 1.56) to C-27 (δ C 178.8) established the

Figure S28 .
Correlation between PTP1B inhibition activities and Glucose uptake stimulation among isolated compounds.FigureS29.LC-MS data of C. operculatus leaves hot water extract and 50%EtOH extract.Table

Table 2 .
Inhibitory activity of isolated compounds against PTP1B enzyme a .
(-) No test.a The values (µM) indicate 50% PTP1B inhibitory effects.These data are expressed as the mean ± S.E.M. of triplicate experiments.b Determined by Lineweaver-Burk plots.c Determined by Dixon plot interpretation.d Positive control.

Table 3 .
Interacting residues and docking scores of 6, 9, 13, and 17 in PTP1B obtained using Libdock Protocol in Discovery Studio.