Alpha-Glucosidase Inhibitory Diterpenes from Euphorbia antiquorum Growing in Vietnam

Bioactive-guided phytochemical investigation of Euphorbia antiquorum L. growing in Vietnam led to the isolation of five ent-atisanes, one seco-ent-atisane, and one lathyrane (ingol-type). The structures were elucidated as ent-1α,3α,16β,17-tetrahydroxyatisane (1), ethyl ent-3,4-seco-4,16β,17-trihydroxyatisane-3-carboxylate (2), ent-atisane-3-oxo-16β,17-acetonide (3), ent-3α-acetoxy-16β,17-dihydroxyatisane (4), ent-16β,17-dihydroxyatisane-3-one (5), calliterpenone (6), and ingol 12-acetate (7). Their chemical structures were unambiguously determined by analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high resolution mass spectrometry, as well as by comparison with literature data. Among them, 1 is a new compound while 2 is an ethylated artifact of ent-3,4-seco-4,16β,17-trihydroxyatisane-3-carboxylic acid, a new compound. Isolates were evaluated for alpha-glucosidase inhibition. Compound 3 showed the most significant inhibitory activity against alpha-glucosidase with an IC50 value of 69.62 µM. Further study on mechanism underlying yeast alpha-glucosidase inhibition indicated that 3 could retard the enzyme function by noncompetitive.

Compounds 1-4, 6, and 7 were evaluated for their alpha-glucosidase inhibition ( Table 1). All tested compounds showed stronger activity than the positive control, acarbose (IC 50 332.5 µM), similar to previously reported ent-atisane [10]. Among them, compound 3 showed the highest alpha-glucosidase inhibition (IC 50 69.62 µM), indicating the important role of the acetonide moiety at C-16 and C-17. Compound 3 was prepared rapidly when 5 reacted with acetone under acidic catalyst at room temperature in one day. This indicated that 3 was an artifact of 5 when using acetone during the isolation.

Alpha-Glucosidase Inhibitory Activity of Isolated Compounds
The in vitro alpha-glucosidase inhibitory activity of 1-4, 6, and 7 was evaluated. All compounds displayed significant alpha-glucosidase inhibitory activity with IC 50 values in the range of 69.62-156.14 µM. The inhibition of isolated compounds on other glycosidases should be evaluated to determine the selectivity. Unfortunately, these tests were not performed due to the minute amounts of isolated compounds.

Inhibition Type and Inhibition Constants of 3 on Alpha-Glucosidase
In order to examine the inhibition mechanism of 3, their activity was measured at the different concentration of 4-nitrophenyl β-D-glucopyranoside (pNPG). The Lineweaver-Burk plots of a kinetic study of 3 ( Figure 4A) showed linearity at each concentration examined (0, 13.8, 27.7, and 55.5 µM), which all intersected the x-axis in the second quadrant. The kinetic analysis revealed that V max decreased while K m remained constant, which showed that 3 acted as a noncompetitive inhibitor. The inhibition constant (K i ) was 65.8 µM ( Figure 4B).

Source of the Plant Material
The aerial parts of E. antiquorum were collected in Binh Thuan province, Vietnam. The scientific name of the plant was determined by Dr. Tran Cong Luan, Faculty of Pharmacy and Nursery, Tay Do University, Can Tho, Vietnam. A voucher specimen of E. antiquorum (No UP B007) was deposited in the herbarium of the Department of Organic Chemistry, Ho Chi Minh City University of Science, National University-HCMC.

Inhibitory Type Assay of 3 on Alpha-Glucosidase
The mechanisms of inhibition of alpha-glucosidase by 3 were determined by Lineweaver-Burk plots (Microsoft Excel 2010

Isolation and Structure Elucidation of the Compounds
Gravity column chromatography was performed on silica gel 60 (0.040-0.063 mm, Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) for checking chromatographic patterns of fractions and isolated compounds was carried out on silica gel 60 F 254 (Merck, Darmstadt Germany) and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Specific rotations were obtained on a Jasco P-1010 polarimeter (Oklahoma City, OK, USA). The HRESIMS were recorded on a MicroOTOF-Q mass spectrometer (Bruker, MA, USA). The NMR spectra were measured on a Bruker Avance 500 MHz spectrometer (Bruker, MA, USA).