Triterpenoids from the Leaves of Diospyros digyna and Their PTP1B Inhibitory Activity

Six new 2α-hydroxy ursane triterpenoids, 3α-cis-p-coumaroyloxy-2α,19α-dihydroxy-12-ursen-28-oic acid (1), 3α-trans-p-coumaroyloxy-2α,19α-dihydroxy-12-ursen-28-oic acid (2), 3α-trans-p-coumaroyloxy-2α-hydroxy-12-ursen-28-oic acid (3), 3β-trans-p-coumaroyloxy-2α-hydroxy-12,20(30)-ursadien-28-oic acid (4), 3β-trans-feruloyloxy-2α-hydroxy-12,20(30)-ursadien-28-oic acid (5), and 3α-trans-feruloyloxy-2α-hydroxy-12,20(30)-ursadien-28-oic acid (6), along with eleven known triterpenoids (7–17), were isolated from the leaves of Diospyros digyna. Their chemical structures were elucidated by comprehensive analysis of UV, IR, HRESIMS, and NMR spectra. All the isolated compounds were evaluated for their PTP1B inhibitory activity. 3β-O-trans-feruloyl-2α-hydroxy-urs-12-en-28-oic acid (13) showed the best inhibition activity with an IC50 value of 10.32 ± 1.21 μM. The molecular docking study found that the binding affinity of compound 13 for PTP1B was comparable to that of oleanolic acid (positive control).


Introduction
Type 2 diabetes (T2DM), a chronic metabolic disease, primarily results from an impaired insulin receptor signaling pathway [1].T2DM accounts for approximately 90% of cases of diabetes.The multifactorial etiology of T2DM, including genetic, environmental, and lifestyle factors, has made its management and prevention a significant challenge for healthcare systems worldwide [2].Protein tyrosine phosphatase 1B (PTP1B), an intracellular enzyme, has been implicated in the negative regulation of insulin signaling [3].The overexpression of PTP1B in various tissues of T2DM patients highlights its pivotal role in the pathogenesis of insulin resistance [4].Recent studies have demonstrated that genetic deletion or the pharmacological inhibition of PTP1B enhances insulin sensitivity and protects against diet-induced obesity [5][6][7][8], thereby underscoring PTP1B as a crucial therapeutical target for T2DM.Several PTP1B inhibitors have shown promise in preclinical models by improving glycemic control and insulin sensitivity [9].Natural products are a rich source of new drug candidates [10] and an important source of PTP1B inhibitors [11,12].

Results
Compound 1 was isolated as a white amorphous powder.Its molecular formula was deduced as C 39 H 54 O 7 based on its HRESIMS ion at m/z 635.3934 [M + H] + .The UV spectrum showed the absorption maxima at 206, 228, and 310 nm.The IR spectrum suggested the presence of hydroxy (3425 cm −1 ), carbonyl (1694 cm −1 ), and aromatic (1605, 1513, and 1454 cm −1 ) groups.The 1 H and 13   1).Comparison of the 1D NMR spectra of 1 with those of 3-O-cis-p-coumaroyltormentic acid [18] indicated similar planar structures, which was further verified by the 2D NMR spectra of 1.
All the isolated compounds were evaluated for their PTP1B inhibitory activity.As a result, compounds 4-6, 10-13, and 15 showed PTP1B inhibition with IC 50 values in the range of 10.32-48.67µM (Table 3), while other compounds were over 50 µM.Compounds 12, 13, and 15 showed better inhibition activity with IC 50 values of 16.20, 10.32, and 17.12 µM, respectively.To know more about the binding and interaction mode between PTP1B and compounds 12, 13, 15, and oleanolic acid, a molecular docking study was conducted by AutoDock Vina.The binding energies of compounds 12, 13, and 15 to PTP1B are −7.1, −7.8, and −7.5 kcal/mol, respectively.Compound 13 is slightly better than that of oleanolic acid (−7.6 kcal/mol), suggesting comparable binding affinity to PTP1B.As shown in Figure 5, these active compounds can dock into the same hydrophobic pocket and bind to the catalytic residues (Gln262, Ala217, and Tyr46) by different interactions as the positive control.

Plant Material
The leaves of Diospyros digyna Jacq.were collected from Zhongshan Haizaoye Agricultural Technology Co., Ltd., Guangdong, China, in July 2018.The plant was identified by Prof. Guangxiong Zhou, College of Pharmacy, Jinan University.A voucher specimen (No.CP2018070903) was deposited in the herbarium of Jinan University.

PTP1B Inhibition Assay
The inhibitory activity of the isolated compounds against PTP1B (Abcam, Cambridge, UK, human recombinant) was assayed according to the method reported previously [28].The reagent p-nitrophenyl phosphate (pNPP) was used as the substrate, and oleanolic acid was used as the positive control.In brief, a 100 µL assay mixture containing 1 µg/mL PTP1B, samples, 4 mM pNPP, 55 mM NaCl, 2.2 mM DTT, 1.1 mM EDTA, and 1 mM BSA in 11 mM Tris-HCl, pH 7.5, was incubated at 37 • C for 30 min in a 96-well plate.The absorbance of 405 nm was measured by a microplate reader.Data were analyzed by GraphPad Prism v.10.2.0 software.All data were obtained in triplicate and presented as means ± SD.

Molecular Docking Analysis
The crystal structure of PTP1B (PDB ID: 8U1E) was obtained from the RCSB Protein Data Bank database.The receptor was prepared by PyMOL 2.5.0 and deposited as a .pdbformat.The 3D structures of the ligands were energy optimized by ChemDraw 3D 18.0 and deposited as a mol2 format.The ligands and receptors for molecular docking analysis were conducted by AutoDock Vina 1.2.2 [29].The grid box parameters (X-center = 0.405, Y-center = 12.132, Z-center = 25.000;x-dimension = 46, y-dimension = 60, z-dimension = 48) were set to cover the binding pocket in the receptor.The docking calculation results were analyzed by PyMOL 2.5.0.
a Overlapped signals were reported without designating multiplicity.
a Positive control.