Chemical constituents from Munronia sinica and their bioactivities

Two new minor constituents, musinisins A (1) and B (2), together with five known compounds (3–7), were isolated from the aerial parts of Munronia sinica. Their structures were established by means of spectroscopic methods and the absolute stereochemistry of 1 was determined by single crystal X-ray experiment. Compound 4 showed antiangiogenic activity evaluated by a zebrafish model and apoptosis-inducing effect on A549 lung cancer cells. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-012-0001-8 and is accessible for authorized users.


Results and Discussion
A 70% aqueous acetone extract prepared from the leaves and stems of M. sinica was partitioned between EtOAc and H 2 O. The EtOAc layer was subjected repeatedly to column chromatography on silica gel, Sephadex LH-20, and RP-18, to afford compounds 1-7. Their structures were shown in Figure  1, and 1 H and 13 C NMR spectroscopic data were listed in Tables 1 and 2.   The anomeric center of the sugar moiety was determined to be -configuration from the large 3 J H1-H2 value (J = 7.8 Hz).
Furthermore, we get a suitable crystal and the X-ray experiment was conducted using an anomalous dispersion with copper radiation (Figure 3), which not only determined the hexose to be D-glucose, but also established the absolute stereochemistry of 1. In combination with IUPAC sequence rule, 15 the configuration of C-3 and C-4 were deduced as R and S, respectively. Thus, the structure of 1 was established as (3R,4S)-3-isopropyl-4-hydroxypentanoic acid 4-O-β-D-glucopyranoside and given the name of musinisin A. The plausible biogenetic pathway of 1, starting from, 3-isopropyl-4methylcyclohexene was also discussed (Scheme 1).
Compound 2 was isolated as yellow powder with [α] 19.9 D -54.86 (c 0.14, MeOH). The molecular formula, C 16 H 24 O 9 , was deduced by HRESIMS ([M + Cl]at m/z 395.1100; calcd 395.1108), with five degrees of unsaturation. The 1 H NMR spectrum (Table 2) showed signals for two methoxyl groups. Comparison of the 1D and 2D NMR data of 2 with those of known compound glucoacetosyringone (3) 10 revealed that compound 2 was structurally similar to 3. The differences were resulted from the appearance of an oxygenated methine signal ( C 68.2, CH;  H 4.6, m), and the lack of a carbonyl signal in 2. This indicated that the carbonyl group at C-7 in 3 was replaced by an oxygenated methine group in 2, which was confirmed by the HMBC correlations of H-2 ( H 6.62), H-6 ( H 6.62) and Me-8 ( H 1.30) with C-7 ( C 68.2) and the 1   value (J = 7.3 Hz). As the aglycone showed a negative optical rotation value that was opposite to that of (1R)-1-phenyl-1propanol, 16 the absolute configuration at C-7 of 2 was suggested to be S. Thus, the structure of 2 was established and given the trivial name musinisin B. The antiangiogenic activities of compounds 1, 2, 4, 5 and 7 were evaluated using a zebrafish model, in terms of the inhibition on the growth of intersegmental vessels, with PTK787 as positive control (IC 50 0.15 g/mL). 17 The results showed that intersegmental vessels of embryos treated with 4 was significantly less than that of the control (0.1% DMSO in sterile salt water). The inhibition ratio of 4 was 58.7% at a concentration of 40 g/mL ( Table 3). The antiproliferative activities of compounds 1-7 were evaluated using A549 lung cancer cells by MTT assay. 18 The results indicated that compounds 1, 2, 4 and 7 showed a certain extent antiproliferative activities (Table 4). From the photos of acridine orange staining, compound 4 showed obvious effect of inducing apoptosis of A549 lung cancer cells (Figure 4).

Experimental Section
General Experimental Procedures. Melting point was obtained on an XRC-1 micro melting point apparatus and is uncorrected. Optical rotations were measured with a Jasco P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Bruker Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker DRX-500 spectrometers. Unless otherwise specified, chemical shifts () were expressed in ppm with reference to the solvent signals. Mass spectra were performed on an API QSTAR Pulsar i spectrometer. Column chromatography was performed with silica gel (200-300 mesh, Qingdao Marine Chemical, Inc., Qingdao, China). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.   Figure 4. Photos of acridine orange staining    Acid Hydrolysis of 2. A solution of 2 (5 mg) in 2 M HCl (3 mL) was heated in a water bath at 70 ˚C for 6 h. After cooling, the reaction mixture was neutralized with NaHCO 3 and extracted with CHCl 3 . Through TLC comparison with authentic sample using CHCl 3 -MeOH (8:2) as a developing system, Dglucose was detected in the water layer (R f = 0.16). The aqueous solution was further concentrated to dryness and subjected to a silica gel chromatography eluting with CHCl 3 -MeOH (9:1) to give D-glucose (1 mg 4 ) to obtain solutions with the test compounds dissolved in 0.1% DMSO. These solutions were aliquot into 96-well plates, and embryos at 24 hpf (hours post fertilization) were also transferred randomly into the above wells. After 24h of treatment, the intersegmental vessels of embryos were visualized with green fluorescent protein labeling and endogenous alkaline phosphatase staining. The antiangiogenic activities of compounds were calculated from the inhibition ratio of angiogenesis. PTK787 was used as the positive control.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-012-0001-8 and is accessible for authorized users.