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

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 embolism¹. 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 cultivars².

The chemical constituents from this genus consisted of diterpenoids, sesquiterpenoids, lignans, coumarins, flavonoids, and some others, of which diterpenoids were the main chemical components with diverse skeletons, including jatropholane, lathyrane, and rhamnofolane type³. Up until recently, more than 65 diterpenoids were identified from this genus⁴. Some of these have been proven to have the effects of antiinflammatory⁵, antiproliferation^{6, 7}, antiplasmodial⁸, antituberculosis⁹, and antitumor^9-11.

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. curcas¹².

Our previous phytochemical investigation on J. curcas from different geographic regions has resulted in the identification of jatropholane and rhamnofolane diterpenes¹³, and of sesquiterpenes¹⁴. 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.

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 (4–10). 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.

Results and Discussion

The CHCl₃-soluble fraction of the methanol extract of the air-dried powdered J. curcas cv. multiflorum was repeatedly chromatographed on silica gel, RP-18, and HPLC to yield ten jatropholane-type diterpenes (1–10). Of them, seven known compounds were identified as sikkimenoid B (4)¹⁵, jatrophaldehyde (5)¹⁶, epi-jatrophaldehyde (6)¹⁶, epi-jatrophol (7)¹⁶, jatrophol (8)¹⁷, jatropholone A (9)¹⁸, and jatropholone B (10)¹⁸ in comparison to their spectroscopic data with those described in the literature.

Compound 1 was assigned the molecular formula C₂₀H₂₄O₃, with nine degrees of unsaturation, on the basis of positive HREIMS at m/z 312.1949 [M]⁺ (calcd for C₂₀H₂₄O₃, 312.1937). The IR spectrum displayed absorption bands for hydroxyl group (3432 cm^-1), carbonyl group (1712 cm^-1), and an exocyclic double bond (1640 and 906 cm^-1). The ¹H and ¹³C NMR spectral data (Table 1) suggests that compound 1 possessed jatropholane-type skeleton comprising a 5/6/7/3 fused ring system, similar to those of jatropholone B (10)¹⁸, except for a hydroxymethyl group in 1 instead of the methyl group in 10. The presence of HMBC correlations from the hydroxymethyl protons at δ_H 5.01 (d, J = 13.5 Hz) and 5.05 (d, J = 13.5 Hz) to the carbon signals at δ_C 134.7 (C-12), 130.0 (C-13), and 153.2 (C-14) indicated that the CH₂OH group was assigned at C-13. The plane structure of 1 could be further confirmed by their COSY and HMBC correlations (Figure 1).

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Compound 1 possessed the same skeleton with the compounds reported in the literature^16-18, the orientations of both H-9 and H-11 in them were α-orientation and confirmed by X-ray crystallography recently¹⁶, thus, the relative configurations of H-9 and H-11 in 1 were α. Moreover, the α-orientation of Me-16 was established by comparison of the chemical shifts, splitting patterns, and coupling constants of Me-16 (δ_C 17.0; δ_H 1.29, d, J = 7.4 Hz) of 1 with those of the literature values (Me-16: δ_C 17.0; δ_H 1.34, d, J = 7.0 Hz) reported for jatropholone B[18a], which also has an α-oriented Me-16 group. Hence, the structure of 1 was deduced and named as jatropholone C.

Compound 2 was isolated as white powder. Its molecular formula was determined to be C₂₁H₂₄O₄ by the molecular ion peak at m/z 340.1679 (calcd for C₂₁H₂₄O₄, 340.1675) in the HREIMS. The ¹H and ¹³C 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) correlations from H-9 (δ_H 1.90), H-11 (δ_H 2.58), Me-20 (δ_H 2.21), and the OMe proton (δ_H 3.74, s) to the ester carbonyl (δ_C 176.2). The 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 β-oriented Me-19 group (δ_C 10.1) in compounds 5a¹⁹. Thus, the -COOMe group in 2 was of α configuration.

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Compound 3 was found by HREIMS (m/z 340.1678 [M]⁺, calcd for C₂₁H₂₄O₄, 340.1675) to have the molecular formula, C₂₁H₂₄O₄, the same as that of 2. Detailed comparison of the ¹H and ¹³C NMR spectroscopic data (Table 1) of compounds 3 and 2 found that the chemical shift values between them were almost the same, with a deviation of ~0.5 ppm, respectively. Nevertheless, the chemical shift of Me-16 (δ_C 17.0) in 3 was remarkably downfield shifted by 1.2 ppm than that of in 2, assuming that they might be C-2 epimers. The β-oriented Me-16 (δ_C 15.8) in 2 was deduced by the similar carbon signal of Me-16 (δ_C 15.0) in 5, and the α-oriented Me-16 (δ_C 17.0) in 3 was determined via the analogous carbon signal of Me-16 (δ_C 16.4) in 6¹⁶. Finally, the structures of compounds 2 and 3 were elucidated and named as jatropholone D and jatropholone E, respectively.

Compounds 1–10 were evaluated for their cytotoxicity against five human tumor cell lines, including HL-60, SMMC-7721, A549, MCF-7 and SW480. However, no cytotoxicity against above assayed cell lines were observed (IC₅₀ > 40 μM). Compounds 4, 6, and 8 demonstrated inhibition activity against the microorganisms with the MIC values from 0.10 to 0.18 mg/mL (Table 2).

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 (δ) were expressed in ppm with respect to the TMS resonance. Semi-preparative HPLC was collected on an Agilent 1100 liquid chromatograph with an YMC-PackProC₁₈RS (YMC, 250 × 10 mm, 5 μm) column. Column chromatography was performed with silica gel (200–300 mesh; Qingdao Marine Chemical Co., Ltd., Qingdao, China), Lichroprep RP-18 silica gel (40–63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (20–150 μm, Pharmacia); Fractions were monitored by TLC under UV light, and spots were visualized by heating silica gel plates sprayed with 10% aqueous H₂SO₄ in EtOH. All solvents including petroleum ether (60–90 ℃) were distilled prior to use.

Plant Material. The plant materials were collected in Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, in July 2012 and identified by Associate Professor Cheng-Yuan Yang. A voucher specimen (No. KIB 20120701) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation. The air-dried roots (4.5 kg) were coarsely powdered and extracted with MeOH (3 × 18 L, each 4 h) under reflux at 55 ℃. The combined MeOH solution was evaporated in vacuo to produce a dark brown residue, which was suspended in water and then extracted with CHCl₃. The CHCl₃-soluble layer (168.5 g) was subjected to column chromatography (CC) over silica gel with acetone/petroleum ether (1:15, 1:10, 1:5, 1:2, 1:1) as elution to afford 5 fractions (Fr.1–Fr.5). Fr.2 (20.0 g) was chromatographed further on RP-18 CC with MeOH/H₂O (55%, 62%, and 75%) to obtain Fr.2.1 (6 g), Fr.2.2 (4 g), and Fr.2.3 (7 g). Fr.2.2 was decolorized on Sephadex LH-20 with MeOH to obtain a brown gum (5.5 g). The gum was further submitted to semipreparative HPLC (3 mL/min, UV detection at λ_max = 254 nm, CH₃CN/H₂O, 60:40) to gain compounds 2 (2.2 mg), 3 (1.7 mg), 5 (20.0 mg), and 6 (23.0 mg). Compounds 9 (32.0 mg) and 10 (28.5 mg) were obtained from fraction Fr.2.2 using a silica gel column (CHCl₃/petroleum ether, 1:5). Fr.2.3 was further treated in sephadex LH-20 with MeOH and semi-preparative HPLC (CH₃OH/H₂O, 70:30) to gain compound 4 (1.5 mg). Fr.3 (18 g, acetone/petroleum ether, 1:5) was fractionated first on RP-18 with a gradient elution with MeOH/H₂O (45%, 60%, and 75%) to yield subfractions Fr.3.1−Fr.3.3. Subsequently, Fr.3.2 (5 g) was further fractionized on a silica gel column eluting with acetone/petroleum ether (1:8) to afford subfractions Fr.3.2.1 (30 mg) and Fr.3.2.2 (240 mg). Fr.3.2.1 was further purified by semi-preparative HPLC (3 mL/min, UV detection at λ_max = 254 nm, CH₃CN/H₂O, 50:50) to yield 1 (1.7 mg). Further separation of Fr.3.2.2 on silica gel eluting with acetone/petroleum ether (1:8, 1:4) gave compounds 7 (20 mg) and 8 (30 mg).

Jatropholone C (1): white amorphous powder; [α]_D ²⁵– 50.5 (c 0.150, CHCl₃); UV (CHCl₃) λ_max (log ε): 314 (3.73), 264 (4.17), 240 (4.26) nm; IR (KBr) ν_max 3432, 1712, 1640, 906 cm^-1; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 335 [M + Na]⁺; HREIMS m/z 312.1949 [M]⁺ (calcd for C₂₀H₂₄O₃, 312.1937).

Jatropholone D (2): white amorphous powder; [α]_D¹⁷ + 15.3 (c 0.150, CHCl₃); UV (CHCl₃) λ_max (log ε): 314 (3.85), 265 (4.28), 240 (4.48) nm; IR (KBr) ν_max 3428, 2955, 2924, 1717, 1696, 1599, 1464 cm^-1; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 363 [M + Na]⁺; HREIMS m/z 340.1679 [M]⁺ (calcd for C₂₁H₂₄O₄ [M]⁺, 340.1675).

Jatropholone E (3): white amorphous powder; [α]_D¹⁷ + 33.7 (c 0.100, CHCl₃); UV (CHCl₃) λ_max (log ε): 313 (3.63), 268 (4.07), 241 (4.26) nm; IR (KBr) ν_max 3422, 2965, 2925, 1638, 1464 cm^-1; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 363 [M + Na]⁺; HREIMS m/z 340.1678 [M]⁺ (calcd for C₂₁H₂₄O₄ [M]⁺, 340.1675).

Cytotoxicity Assay. Compounds 1–10 were evaluated for their cytotoxicity against several human tumor cell lines, including breast cancer MCF-7, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, lung cancer A-549, and colon cancer SW480 by the MTT method¹³ as previously reported.

Antibacterial Assays. The microorganisms employed in the assay were: S. aureus (ATCC 25923), E. coli (ATCC 25922), B. subtilis (ATCC 11060), P. aeruginosa (CMCC 10104), and S. epidermidis (CMCC 26069). To the well-prepared agar plates, microbial cells along with the compounds were suspended in Mueller Hinton broth at the density of 10⁸ CFU/mL and incubated at 37 ℃ 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 ℃. 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 plates²⁰.

Notes

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.

Acknowledgments

The authors are sincerely grateful to Prof. Cheng-Yuan Yang of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, for the identification of the plant. This work was financially supported by the Key Projects of the Chinese Ministry of Science and Technology (2007BAD32B01-03 and SB2007FY400), the National Natural Science Foundation of China (81202437) and the National Knowledge Innovation Program of Chinese Academy of Sciences (KSCX2-YW-G-038).