Glucose Uptake Stimulatory and PTP1B Inhibitory Activities of Pimarane Diterpenes from Orthosiphon stamineus Benth

Seven pimarane diterpenes (1–7) were isolated from Orthosiphon stamineus Benth. by assay-guided isolation. All of the isolates possessed a 2-deoxy-2-((7-nitro-2,1,3-benzoxadiazol-4-yl)amino)-d-glucose uptake effect in 3T3-L1 adipocytes at concentrations of 5 and 10 μM. Most of them showed potent inhibition against protein tyrosine phosphatase 1B with IC50 values ranging from 0.33 to 9.84 μM. In the kinetic study, all inhibition types were exposed for the examined potencies, including mixed-competitive (1), non-competitives (3 and 5), competitive (6), and uncompetitive (7). The results suggested that O. stamineus and its pimarane diterpenes might exert the hypoglycemic effect via the insulin signaling pathway targeting inhibition of protein tyrosine phosphatase 1B (PTP1B) activity.


Cell Viability Assay
The viability of cultured cells was assessed using an MTT assay. In brief, 3T3-L1 (1 × 10 4 cells/well) cells seeded in 96-well plates were treated with various concentrations of samples for 48 h. Then, MTT solution (1 mg/mL) was added to each well, and the cells were incubated at 37 °C for 1 h. Finally, DMSO was added to dissolve the formazan crystals. Absorbance was measured at 540 nm using a spectrophotometer (Immuno Mini NJ-2300, Japan).

Adipocyte-Based Measurement of 2-NBDG Uptake
3T3-L1 adipocytes grown in black 96-well plates were incubated with each sample for 24 h at 37 °C in a 5% CO2 atmosphere. Subsequently, 250 µM 2-NBDG (dissolved in PBS with 1% BSA) was added to the cells and incubated for a further 30 min. After incubation, cells were washed two times with PBS to remove excess fluorescence in the wells. Then, fluorescence retained by the cells was measured using a PerkinElmer Victor3 V 1420 Multilabel Plate Counter at an excitation and emission wavelength of 485 nm and 535 nm, respectively.

PTP1B Inhibition Assay
Protein tyrosine phosphatase 1B (human recombinant) was purchased from Biomol International LP, Plymouth Meeting, PA, USA, and the inhibitory activities of the tested samples were evaluated using the method as described in [31]. Briefly, 0.05−0.1 µg of PTP1B (BIOMOL International L.P., Plymouth Meeting, PA, USA) and 4 mM p-NPP in a buffer containing 1 mMdithiothreitol, 0.1 M NaCl, 1 mM EDTA, and 50 mM citrate (pH 6.0), with or without test compounds, were added as 100 µL of a final volume to each of the 96 wells. After the reaction mixture was incubated at 37 °C for 30 min, 10 M NaOH was added to quench the reaction. PTP1B enzyme activity was determined by the amount of produced p-nitrophenol at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control, which did not contain the PTP1B enzyme.

Determination of the Inhibition Mode of Active Compounds
The Lineweaver-Burk plot and Dixon plot experiments were carried out in the presence and absence of the inhibitors with various concentrations of p-NPP as the substrate. The inhibition modes of the tested compounds were assessed on the basis of their inhibitory effects on the Km (dissociation constant) and Vmax (maximum reaction velocity) of the enzyme, which were determined by the Lineweaver-Burk plot experiment. The Lineweaver-Burk plot is the double reciprocal plot of the enzyme reaction velocity (V) versus the substrate (p-NPP) concentration (1/V versus 1/(p-NPP)).

Statistical Analysis
Data are represented as means ± SD of at least three independent experiments performed in triplicated assays and determined by regression analysis. For statistical analysis of the data for single comparison, the significance between means was determined by the Student's t test. Sigma Plot program version 11.0 was used for analysis of the kinetic data.