A New Dammarane-type Triterpene with PTP1B Inhibitory Activity from Gynostemma pentaphyllum

Results and Discussion

Compound 1 was obtained as white solid. Its molecular formula was determined as C30H45O3 by HRESIMS. The 1H NMR spectrum of 1 showed the presence of six methyl singlets at δ 0.91, 0.92, 0.97, 1.02, 1.06, 1.91, an oxymethine signal as a doublet of doublets at d 4.75 (J = 12.4, 3.6 Hz), a exomethyene singlets at δ 5.22 and 5.25, and another olefinic signal at δ 6.59 as a broad triplet (J = 6.4 Hz) (Table 1). These data indicated that the structure of 1 might contain terminal olefinic and tri-substituted olefinic groups, which also supported by the presence of four sp2 signals (δ 149.4, 139.4, 128.6 and 113.7) in 13C NMR spectrum of 1 (Table 1). Moreover, the 13C NMR spectrum displayed signals attributed to an oxymethine carbon (δ 81.1), an ester carbonyl group (δ 166.3) and a ketone carbonyl group (δ 218.3). In addition, six methyls, ten methylenes, six methines, and eight quaternary carbons were determined by display of its DEPT-135 data. All the above observations and chemical shifts suggested that 1 could be a dammarane-type triterpene. In particular, the 1H and 13C NMR spectra of 1 were in close agreement with those of 24(E/Z)-3-oxodammara-20,24-dien-26- al, except for the side-chain moiety.9 The presence of an α,β- unsaturated-δ-lactone moiety was established by observation of the HMBC spectrum data (Fig. 2), which showed long-range correlations from δ 4.75 (H-22) to δ 149.4 (C-20) and 113.7 (C-21), from δ 6.59 (H-24) to δ 81.1 (C-22), 29.2 (C-23), 128.6 (C-25), and 166.3 (C-26), from δ 5.22, 5.25 (H-21) to δ 149.4 (C-20) and 81.1 (C-22). The relative stereochemistry of the basic ring moiety was assigned by comparison of chemical shifts with related dammarane-type triterpenes.10 The δ-lactone moiety was confirmed the 22S configuration on the basis of a negative Cotton effect at 257 nm (Δε −16.9) in the CD spectrum.11 Therefore, the structure of 1 was determined as 22(S)-3-oxodammar-20,24-dien- 26,22-lactone.

Figure 2.Key HMBC correlations of compound 1.

Table 1.aChemical shifts in ppm relative to TMS; coupling constants (J) in Hz.

The known compounds 2-7 were identified as 24-(Z)-3- oxodammar-20(21),24-dien-27-oic acid (2),12 25-methoxy- 5α-dammar-20-en-3β,24-diol (3),13 24(S)-25-epoxy-5α-protost- 20,25-dien-3-one (4),13 (20S,23S)-3β,20-dihydroxyldammarane- 24-ene-21-oic acid-21,23-lactone (5),14 20(S)- dammarane-25(26)-ene-3β,12β,20-triol (6),15 (20S,24S)- dammarane-25(26)-ene-3β,12β,20,24-tetrol (7)15 based on the NMR data.

All the isolates were assayed their inhibitory activity against PTP1B using an in vitro assay (Table 2), and RK-682 was used as positive control.16 As shown in Table 2, compounds 1, 2 and 4 showed potential inhibitory activities of PTP1B with IC50 values ranging from 13.2 ± 1.9 to 19.2 ± 2.1 μM, while the remaining compounds displayed moderate effects. The structure-activity relationships of 1-5 against PTP1B indicated that the presence of a hydroxyl group located at C-3 might be responsible for the decrease of inhibitory activity of these compounds. Among the above isolates, compounds 3-4 were reported for the first time from this plant.

Table 2.aIC50 values were determined by regression analyses and expressed as mean ± SD of three replicates. bPositive control.16

 

Experimental

General Experimental Procedures. UV spectra were taken in MeOH using a Shimadzu spectrophotometer. Nuclear magnetic resonance (NMR) spectra were obtained from a Varian Unity Inova 400 MHz spectrometer using TMS as the internal standard. All accurate mass experiments were performed on a Micromass QTOF (Micromass, UK) mass spectrometer. Column chromatography was conducted using silica gel 60, Sephadex LH-20 and RP-18 for thin-layer chromatography, precoated TLC silica gel 60 F254 plates from Merck were used. HPLC runs were carried out using a Shimadzu System LC-10AD pump equipped with a model SPD-10Avp UV detector, and an Optima Pak® C18 column (10 × 250 mm, 10 mm particle size, RS Tech Korea).

Plant Material. The root of G. pentaphyllum was collected in Xuzhou, Jiangsu province, People’s Republic of China, and authenticated by Professor Gao Li (College of Pharmacy, Yanbian University). A voucher specimen of the plant (No. 20101006) was deposited at the College of Pharmacy, Beihua University, Jilin, China.

Extraction and Isolation. The root (5.0 kg) of G. pentaphyllum was extracted with MeOH at room temperature for 2 weeks and the solution was concentrated to obtain a crude extract. This extract was suspended in H2O, partitioned successively with CHCl3 and EtOAc, and then the organic solvents were removed. A portion of the CHCl3-soluble fraction (10.0 g) was chromatographed over a silica gel column using a gradient of CHCl3-MeOH (from 70:1, 50:1, 20 :1 to 10:1), and was separated into 10 fractions (Fr.D1- Fr.D10). Fr.D4 (CHCl3-MeOH 10:1, 1.0 g) was chromatographed over silica gel, eluted with a stepwise gradient of n-hexane- EtOAc (from 20:1, 19:1 to 0:1) to afford 10 subfractions (Fr.D4.1-Fr.D4.10). Purification of Fr.D4.4 (110.0 mg) by semipreparative HPLC using an isocratic solvent system of 95% MeCN in H2O over 60 min to yield compounds 1 (4.4 mg) and 2 (6.4 mg). The EtOAc extract (50.7 g) was subjected to silica gel CC and eluted with a gradient of CH2Cl2/MeOH (25:1, 20:1, 15:1, 10:1, to 5:1) to yield 5 fractions (Fr.E1-E5). Fr.E2 (709.0 mg) was purified by preparative HPLC using an isocratic solvent system of 75% MeCN in H2O over 30 min followed by 80% MeCN in H2O over 70 min to obtain compounds 5 (5.9 mg) and 6 (5.1 mg). Fr.E3 (2.2 g) was subjected to an RP-18 column and was eluted with MeOH-H2O (1:1, 2:1, to 10:1) to yield six fractions (Fr.E3.1-Fr.E3.6). The most active fraction, Fr.E3.5 (571.0 mg), was further separated by a silica gel column eluted with CHCl3-MeOH (40:1, 35:1, to 10:1) to yield 7 subfractions (Fr.E3.5.1-Fr.E3.5.7). Fr.E3.5.3 was purified by preparative HPLC using an isocratic solvent system of 50% MeCN in H2O over 50 min obtain compound 3 (3.4 mg). Fr.E3.5.5 was separated by HPLC, using a gradient of 40- 50% MeCN in H2O as the mobile phase to produce compounds 7 (3.1 mg) and 4 (4.9 mg).

Compound 1: White solid; [α]D +30° (c, 0.08, CHCl3); 1H NMR (400 MHz, CDCl3) and 13C NMR data (100 MHz, CDCl3) spectral data see Table 1; HRESIMS m/z 453.3363 [M + H]+ (Δ −1.5 mmu, calcd for C30H45O3).

PTP1B Assay. The enzyme activity was measured using p-nitrophenyl phosphate (pNPP) as described previously.16 To Each 96 well (final volume: 100 μL) was added 2 mM pNPP and PTP1B (0.05-0.1 μg) in a buffer containing 50 mM citrate (pH 6.0), 0.1 M NaCl, 1 mM EDTA, and 1 mM dithiothreitol (DTT), with or without test compounds. Following incubation at 37 °C for 30 min, the reaction was terminated with 1 M NaOH. The amount of produced p-nitrophenol was estimated by measuring the absorbance at 405 nm.