New jatropholane-type diterpenes from Jatropha curcas cv. Multiflorum CY Yang

Three new jatropholane-type diterpenoids, jatropholones C-E (1–3), along with seven other known compounds, including sikkimenoid B (4), jatrophaldehyde (5), epi-jatrophaldehyde (6), epi-jatrophol (7), jatrophol (8), jatropholone A (9), and jatropholone B (10), were isolated from the roots of a natural cultivar of Jatropha curcas (J. curcas cv. Multiflorum CY Yang). The structural elucidations of 1–3 were accomplished by extensive NMR analysis. Compounds 4, 6, and 8 demonstrated inhibition activity against the microorganisms with the MIC values from 0.10 to 0.18 mg/mL. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s13659-013-0031-x and is accessible for authorized users.


Introduction
The genus Jatropha belonging to the family Euphorbiaceae contains a number of traditionally important medicinal plants. Approximately 170 species are found in tropical Asia and Africa, while only three common cultivated and three wild species (J. curcas, J. podagrica, and J. multifida), together with two cultivated species (J. gossypifolia and J. integerrima) distributed in China. J. curcas and J. podagrica, have been used as Chinese traditional medicines for the treatment of injuries swelling, fracture, skin itching, eczema, and acute gastroenteritis embolism 1 . Recently, two natural cultivars of J. curcas (J. curcas cv. Multiflorum CY Yang and J. nigroviensrugosus CY Yang) were discovered in the Yuanjiang River, Yunnan province and had been registered as new cultivars 2 .
J. curcas cv. Multiflorum, as one of two natural cultivars of J. curcas, had the morphological characteristics as follows: I) It can blossom many times in a year with a number of flowers, fruits, and branches; II) It can blossom at the beginning of germination after cutting branches; III) Compared with J. curcas, the most striking trait is that the production of seed of the plant is higher than that of J. curcas 12 .
Our previous phytochemical investigation on J. curcas from different geographic regions has resulted in the identification of jatropholane and rhamnofolane diterpenes 13 , and of sesquiterpenes 14 . However to date, no phytochemical research has been carried out on J. curcas cv. Multiflorum. Therefore, in order to identify the chemical components and examine for structurally diverse and biologically significant compounds, an initial investigation on the chemical compositions of J. curcas cv. Multiflorum was undertaken.
*To whom correspondence should be addressed. E-mail: mhchiu@mail.kib.ac.cn Our first-time phytochemical study on the plant collected in Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, led to the isolation of three new jatropholane-type diterpenoids (1-3), along with seven known jatropholane analogs (4)(5)(6)(7)(8)(9)(10). Their structures were determined by analysis of the spectroscopic data. Compounds 1-10 had been evaluated for their cytotoxic activity and compounds 4, 6, 8, and 10 were further tested for their antibacterial effect against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus epidermidis. Herein, the isolation, structural elucidation and biological evaluation of these compounds were reported.
Compound 2 was isolated as white powder. Its molecular formula was determined to be C 21 H 24 O 4 by the molecular ion peak at m/z 340.1679 (calcd for C 21 H 24 O 4 , 340.1675) in the HREIMS. The 1 H and 13 C NMR data (Table 1) of compound 2 bore a close resemblance to those of jatrophaldehyde (5) 16 , and the distinction was that the aldehyde group at C-18 in 5 was replaced by a methoxycarbonyl group in 2. This deduction was further confirmed by the HMBC (Figure 2)   distinct correlations of Me-18 with H-9 and H-11 were observed in the ROESY spectrum of 1. However, the abovementioned correlations were not found in the ROESY spectrum of 2. Additionally, the carbon resonance of Me-19 in 2 was δ C 10.3, which was similar to that of -oriented Me-19 group (δ C 10.1) in compounds 5a 19 . Thus, the -COOMe group in 2 was of  configuration.  16 . Finally, the structures of compounds 2 and 3 were elucidated and named as jatropholone D and jatropholone E, respectively.

Experimental Section
General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were obtained in MeOH with a Shimadzu UV-2401 PC spectrophotometer. IR spectra were recorded on a Brucker Tensor 27 infrared spectrophotometer with KBr pellets. ESIMS spectra were performed on an API QSTAR Pulsar spectrometer, while HREIMS were measured on a Waters Autospec Premier P776 mass spectrometer instrument in positive ion mode. 1D and 2D NMR spectra were carried out on Bruker Avance III-600MHz spectrometer. Chemical shifts () were expressed in ppm with respect to the TMS resonance.   (CMCC 26069). To the well-prepared agar plates, microbial cells along with the compounds were suspended in Mueller Hinton broth at the density of 10 8 CFU/mL and incubated at 37 °C for 18 h under aerobic conditions. The compounds were dissolved in DMSO to give six different concentrations of 1 µg/mL, 3 µg/mL, 9 µg/mL, 27 µg/mL, 81 µg/mL and 243 µg/mL, added 200 µL to oxford cup with the diameter of 8 mm. The blank controls of microbial culture were incubated with limited DMSO under the same conditions. The DMSO was determined not to be toxic at a limited amount under the experimental conditions. The controls comprised the inoculated growth medium without test agents, and sample blanks in growth medium only. The plates were incubated for 24 h at 37 °C. The data was obtained through determining the inhibition zone diameters with oxford cup. The MIC value of each compound was determined using a microdilution assay in sterile 96-well microtiter plates 20 .

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0031-x and is accessible for authorized users.