Three New Myrsinol Diterpenes from Euphorbia prolifera and Their Neuroprotective Activities

Three new myrsinol diterpenes were isolated from the roots of Euphorbia prolifera. Their structures were elucidated as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-O-acetyl-14α-O-benzoyl-10,18-dihydromyrsinol (1), 2α-O-isobutyryl-3β-O-propion-yl-5α,7β,10,15β-tetra-O-acetyl-10,18-dihydromyrsinol (2), and 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol (3) on the basis of spectroscopic data analyses (IR, ESI-MS, HR-ESI-MS, and 1D and 2D NMR). Their neuroprotective activities were evaluated and compounds 1 and 2 showed neuroprotective effects against MPP+-induced neuronal cell death in SH-SY5Y cells.


Results and Discussion
Compound 1 was isolated as colorless needles. Its molecular formula was determined as C 41 (Tables 1 and 2) was also deduced based on those reported diterpenes with isobutyryl groups from the genus Euphorbia [4,5,7]. Apart from the above 21 signals for the substituents (five acetoxy groups, one isobutyryloxy group, and one benzoyloxy group), there are additional 20 resonances exhibited for the parent skeleton in the 13 C-NMR spectrum, which comprised four methyls (C-16, C-18, C-19, and C-20), two methylenes (C-1 and C-17), nine methines (C-3, C-4, C-5, C-7, C-8, C-9, C-11, C-12, and C-14), and five quaternary carbons (C-2, C-6, C-10, C-13, and C-15). By comparing the chemical shifts of C-1C-20 of compound 1 with those of myrsinol diterpenes reported in the literature [4,5,7], the presence of the 10,18-dihydromyrsinol skeleton was obvious. In order to confirm the 10,18-dihydromyrsinol skeleton and determine the positions of the acyloxy groups, the following HMQC and HMBC experiments were performed. By the interpretation of 1D and 2D NMR spectra, the 10,18-dihydromyrsinol skeleton was defined ( Figure 1 the methyl protons (δ H 2.13) of the C-10 acetoxy group, implied that the two acetoxy groups were located at C-15 and C-10, respectively. Consequently, the isobutyryloxy group could be assigned only to C-2 based on the chemical shift for C-2. By further analyzing the HMQC, HMBC, and 1 H-1 H COSY spectra (Figure 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of 1 was established.  The relative configuration of 1 was elucidated based on the NOESY spectrum and the comparison of NMR data with those of reported myrsinol diterpenes in the literature [4,5,7,13,14]. For all of the reported natural myrsinol diterpenes, the three rings (5/7/6) comprising the myrsinol-skeleton are trans-fused with each other, H 3 -16, H-12, the side chain at C-11, and the acyloxy group at C-15 are biogenetically -oriented, and H-4 and H 2 -17 are biogenetically -oriented [7,13]. NOESY  [7,13]. Therefore, the structure of compound 1 was characterized as 2α-O-isobutyryl-3β,5α,7β,10,15β-penta-  14 Na 717.3098). The 1 H-and 13 C-NMR spectra of 2 were very similar to those of compound 1, which implied that compound 2 should also be a 10,18-dihydromyrsinol diterpene. Close similarities of the chemical shifts from C-1 to C-20 in 2 with those in 1 ( Table 2) suggested that compounds 2 and 1 had the same parent skeleton [4,5,7]. In addition to the 20 skeletal carbons, the 13 C-NMR spectrum revealed additional 15 carbons for the substituent groups, which were deduced and defined as four acetoxy groups, one isobutyryloxy group, and one propinoyloxy group based on the analyses of HMQC and HMBC spectroscopic data of 2 [4,5,7]. The locations of the six acyloxy groups were elucidated by the interpretation of the HMBC spectrum as in the case of 1. By the HMBC correlations of the protons H-3 (δ H 5.22), H-5 (δ H 5.83), and H-7 (δ H 4.79), with the corresponding carbonyl signals at δ C 173.4, 169.1, and 170.2, the two acetoxy groups and the propinoyloxy group were attributed to C-5, C-7, and C-3, respectively. There were no long-range correlations observed for H-14 (δ H 4.05) to the carbonyl carbons of the acyloxy groups, which indicated that the substituent at C-14 was a hydroxy group based on the chemical shift for C-14. The remaining two acetoxy groups and one isobutyryl group were assigned to C-10, C-15, and C-2, respectively, which was supported by the NOESY correlation of the H-14 (δ H 4.05) and H-1 (δ H 2.13) to the methyl protons (δ H 1.91) of the C-15 acetoxy group and the H 3 -19 (δ H 1.48) to the methyl protons (δ H 2.04) of the C-10 acetoxy group. The similar myrsinol-type diterpene skeleton of 2 compared to those of compound 1 implied the same trans-fusion of the three rings (5/7/6) [7,13]. The  15 Na 841.3047), which was compatible with the NMR data. On comparing the chemical shifts for the skeletal carbons in 3 with those for C-1C-20 in compound 1, the almost coincidence implied that compounds 3 and 1 had the same characteristic 10,18-dihydromyrsinol skeleton [4,5,7]. The only difference between 3 and 1 was that the one isobutyryloxy group in 1 was replaced by a benzoyloxy group in 3, which was deduced based on the 13 C-NMR spectrum and the chemical shifts for the benzoyloxy groups in the reported myrsinol diterpenes [7,13]. Following the same NMR procedures used for 1, the locations of the acyloxy groups in 3 were determined by the analysis of the HMBC spectrum, which revealed that the five acetoxy groups were attached at C-3, C-5, C-7, C-10, and C-15, and the two benzoyloxy groups were at C-2 and C-14, respectively. The same relative configuration to be inferred for compound 3 and compound 1 was revealed by the careful comparison of the NOESY spectra of 3 and 1. The structure of compound 3 was therefore characterized as 2α,14α-di-O-benzoyl-3β,5α,7β,10,15β-penta-O-acetyl-10,18-dihydromyrsinol.  In order to explore the potential biological activities of these new diterpenes isolated from the roots of E. prolifera, the neuroprotective effects against MPP + -induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells were evaluated as described previously [11,15]. Bakkenolide H was used as a positive control [16]. Compounds 1 and 2 exhibited neuroprotective activities against MPP + -induced neuronal cell death in SH-SY5Y cells. The neuroprotective effects of the two compounds were shown in Figure 3. MTT assay indicated that compounds 1 and 2 (3-30 μM) neither affected the cell viability nor showed significant cytotoxicity with the absence of MPP + (data not shown). Compound 3 was not investigated for the neuroprotective effects because of inadequate amount.

General Experimental Procedures
The IR spectra were taken on a on a Bruker Tensor 27 FT-IR spectrometer with KBr discs. Optical rotations were measured in CH 2 Cl 2 using an Autopol IV automatic polarimeter. The ESI-MS spectra were obtained on a LCQ-Advantage mass spectrometer made by Finnigan Company of America. HR-ESI-MS spectra were obtained using an Ionspec 7.0 T FTICR MS. 1D and 2D NMR spectra were recorded on a Bruker AV 400 instrument (400 MHz for 1 H and 100 MHz for 13 C) with TMS as an internal standard. HPLC separations were performed on a CXTH system (Beijing Chuangxintongheng instruments Co. Ltd., China), equipped with a UV3000 detector at 210 nm, and a YMC-pack ODS-AM (250 × 20 mm) column. Silica gel (200-300 mesh, Qingdao Marine Chemical Group Co. Ltd., China) was used for column chromatography. Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Co. Ltd., China. Biological reagents were from Sigma Company. Human dopaminergic SH-SY5Y cells were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). Biological reagents were from Sigma Company.

Plant Material
The roots of Euphorbia prolifera Buch-Ham were collected from Kunming, Yunnan Province, China, in July 2010. The botanical identification was made by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China), and a voucher specimen (No. 20100705) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China.

Extraction and Isolation
The air-dried roots of E. prolifera (3.8 kg) were powdered and extracted with MeOH (3 × 20 L) under reflux. The solvent was evaporated to obtain a crude extract (900 g). The extract was suspended in H 2 O (0.9 L) and partitioned with EtOAc (3 × 0.9 L) to afford the EtOAc soluble part after removing the solvents. The EtOAc soluble part (150.0 g) was subjected to a silica gel column chromatography using a gradient solvent system from 140% acetone in petroleum ether to give eight fractions (F 1 F 8 ) based on TLC analyses. Fraction F 8 was fractionated by MPLC over ODS eluting with a step gradient from 60% to 90% MeOH in H 2 O to obtain four subfractions (F 8-1 F 8-4 ). Subfraction F 8-3 was purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 73% MeOH in H 2 O) to afford compound 1 (16.5 mg). The further purification of F 8-4 with the same HPLC system using 78% MeOH in H 2 O, resulted in the isolation of compound 3 (3.2 mg). Fraction F 7 , using the same MPLC (6090% MeOH in H 2 O), provided four subfractions F 7-1 F 7-4 , and the further purification of F 7-2 by the above HPLC system (70% MeOH in H 2 O) afforded compound 2 (11.9 mg).  Table 2.

Bioassay for Neuroprotective Activity
Human dopaminergic neuroblastoma SH-SY5Y cells were cultured at 37 °C in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/mL of penicillin/streptomycin in a water-saturated atmosphere of 95% air and 5% CO 2 . The cells were disassociated by incubation with 1 mM of ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in phosphate-buffered saline (PBS) for 15 min and then seeded in 96-well culture plates (1 × 10 4 cells/well). The cells were incubated at 37 °C in a 5% CO 2 humidified air incubator for 24 h. The cells were pre-treated for 2 h with various concentrations (3, 10 and 30 μM) of the compounds before incubation in a medium containing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP + ). MTT dissolved in phosphate-buffered saline was added at the end of incubation to a final concentration of 0.5 mg/mL. After incubation for 4 h at 37 °C and 5% CO 2 , the supernatant was removed and the formed formazan crystals in the viable cells were measured at 490 nm by using a microplate reader (Thermo Fisher Scientific Inc., USA). Experiments were carried out in triplicate. A statistical analysis was performed by a one-way analysis of variance (ANOVA) followed by post hoc multiple comparisons using the Student-Newman-Keuls method. Data are expressed as the mean ± SD of three assays.

Conclusions
In summary, three new myrsinol diterpenes were successfully isolated from the roots of E. prolifera. Their structures were elucidated on the basis of extensive spectroscopic data analyses. Biological evaluation disclosed that two of them exhibited strong neuroprotective effects against MPP + -induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells. The results of our chemical investigation further revealed the chemical constituents of E. prolifera. While, the current biological data suggest that myrsinol diterpenes with strong neuroprotective activities may probably be useful for the treatment of nervous system diseases, such as Parkinson's disease [12]. Further biological studies on these compounds are still underway by our group.