Chinese Journal of Natural Medicines  2019, Vol. 17Issue (4): 298-302  
0

Cite this article as: 

CHEN Lin, HUANG Jia-Luo, ZHANG Lei, TIAN Hai-Yan, YIN Sheng. Jatrogricaine A: a new diterpenoid with a 5/6/6/4 carbon ring system from the stems of Jatropha podagrica[J]. Chinese Journal of Natural Medicines, 2019, 17(4): 298-302.
[Copy]

Research funding

This work was supported by the National Natural Science Foundation of China (Nos. 81573302 and 81722042), the Science and Technology Planning Project of Guangdong Province (No. 2015A020211007), and the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306047)

Corresponding author

YIN Sheng, E-mail: yinsh2@mail.sysu.edu.cn

Article history

Received on: 18-Oct-2018
Available online: 20 April, 2019
Jatrogricaine A: a new diterpenoid with a 5/6/6/4 carbon ring system from the stems of Jatropha podagrica
CHEN Lin1 , HUANG Jia-Luo1 , ZHANG Lei1 , TIAN Hai-Yan2 , YIN Sheng1     
1 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China;
2 Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China
[Abstract]: Jatrogricaine A (1), a new diterpenoid possessing a 5/6/6/4 carbon ring system, together with eight known diterpenoids (2-9) were isolated from the stems of Jatropha podagrica. Their structures were elucidated by extensive spectroscopic methods and the absolute configuration of 1 was determined by single crystal X-ray diffraction analysis. All compounds were evaluated for their anti-inflammatory activities in vitro, and compound 3 showed significant inhibitory effects against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells with an IC50 of 13.44 ±0.28 μmol·L-1, being comparable to the positive control, quercetin (IC50 17.00 ±2.10 μmol·L-1).
[Key words]: Jatropha podagrica     Euphorbiaceae     Diterpenoids     Anti-inflammatory activity    
Introduction

The genus Jatropha (Euphorbiaceae) comprises approximately 175 species mainly growing in the tropic and subtropical areas of Africa, Asia, and Latin America [1]. Previously phytochemical investigations of this genus revealed that it was a rich resource of structurally intriguing macrocycle diterpenoids, such as tiglianes, daphnanes, lathyreanes, and rhamnofolanes [2-5]. These diverse secondary metabolites exhibited various biological activities, such as cytotoxicity, antibacterial, and anti-inflammatory [5-6].

Jatropha podagrica Hook is a shrub widely cultivated as an ornamental plant in tropical countries. The seed oil of J. podagrica is used as a folk medicine by Nigerian for the treatment of rheumatic condition, itch, parasitic skin diseases, and fever [7-8]. Previous chemical investigation of this plant led to the isolation of several diterpenoids [6, 9] and alkaloids [7-8], some of which showed significant antibacterial, neuromuscular-blocking and hypotensive activities. As a part of our effort to discover structurally interesting and biologically active secondary metabolites from genus Jatropha [10-12], a new polycyclic diterpenoid with a rare 5/6/6/4 carbon ring system, along with eight known diterpenoids, were isolated from the stems of J. podagrica. All the isolates were tested for their inhibitory activities against NO production in LPS-induced RAW264.7 macrophage cells, and compound 3 exhibited significant activity. Herein, we report the isolation, structural elucidation, and bioactivity evaluation of compounds 1-9.

Results and Discussion

The air-dried powder of the stems of J. podagrica was extracted with 95% EtOH at room temperature. The concentrated extract was then suspended in H2O and successively partitioned with petroleum ether and EtOAc. Compounds 1-9 were isolated from the EtOAc fraction by various column chromatographic methods (Fig. 1).

Fig. 1 The structures of compounds 1-9

Compound 1 was obtained as colorless crystals. The molecular formula C20H28O4 was established by its HR-ESIMS data at m/z 355.1880 [M + Na]+ (Calcd. 355.1880), indicating seven degrees of unsaturation (DOUs). The IR spectrum displayed absorption bands for hydroxyls (3553 and 3408 cm-1) and carbonyl group (1702 cm-1). The 1H NMR spectrum showed four singlet methyls [dH 1.17, 1.12, 1.08, and 0.99 (each 3H)], a doublet methyl [δH 1.26 (3H, d, J = 7.5 Hz)], two oxygenated methines [δH 4.73 (1H, t, J = 3.0 Hz) and 3.50 (1H, d, J =8.7 Hz)], and a series of aliphatic multiplets. The 13C NMR combined with DEPT experiment resolved 20 carbon signals consistent with five methyls, three methylenes, five methines (two oxygenated), a tetrasubstituted double bond (δC 157.6 and 147.6), two carbonyl groups (δC 214.8 and 204.9), and three sp3 quaternary carbons. As three of the seven DOUs were attributed to one double bond and two carbonyl groups, the remaining four DOUs required that 1 was tetracyclic. Above information was very similar to those of euphoractine T [13], a known diterpene with a 5/6/6/4 carbon ring system, except for the replacement of an oxymethine in euphoractine T by a methylene in 1. Combined with the upfield-shifted chemical shift of C-2 in 1 with respect to that in euphoractine T (δC 41.5 in 1; δC 51.1 in euphoractine T), 1 was assigned as 1-dehydroxylated derivative of euphoractine T.

The planar structure of 1 was further supported by the detailed interpretation of 2D NMR spectra. Two spin systems as depicted in Fig. 2 were assigned by the 1H-1H COSY of H-1α/H-2/H3-16 and H2-7/H2-8/H-9/H-12/H-11. These fragments, quaternary carbons, double bonds, and carbonyls were further linked by HMBC correlations from H3-16 to C-1/C-2/ C-3, H3-17 to C-5/C-6/C-7/C-13, H3-18/19 to C-9/C-10/C-11, and H3-20 to C-6/C-12/C-13/C-14, which generated a 5/6/6/4 carbon ring system.

Fig. 2 1H-1H COSY (━) and key HMBC (→) correlations of 1

The relative configuration of 1 was established by the NOESY experiment. The NOE correlations (Fig. 3) of H-12/H3-17 and H3-19 indicated these protons were on the same side and were arbitrarily assigned as β-orientation. Thus, the NOE correlations of H-11/H-9, H3-20, and H3-18 as well as H3-20/H-5 assigned that those protons are α-oriented. The relative configuration of C-2 could not be assigned due to the long distance between C-2 and other chiral centers. Fortunately, the crystals of 1 were obtained from a mixed solution of CH2Cl2-MeOH (1 : 5) and single-crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation (Fig. 4) was performed. The final refinement with a Flack parameter of -0.14 (13) permitted the establishment of the absolute configuration of 1 as 2S, 5R, 6S, 9S, 11R, 12R, 13S. Compound 1 was given a trivial name jatrogricaine A.

Fig. 3 Key NOE correlations of 1
Fig. 4 X-ray crystal structure of 1

The known compounds, 15-epi-4E-jatrogrossidentadion (2) [14], 4E-jatrogrossidentadion (3) [14], 2-hydroxyisojatrogrossidion (4) [14], 2-epi-hydroxyisojatrogrossidion (5) [14], 4Z-jatrogrossidion (6) [14], jatrocurcasenone D (7) [15], and sikkimenoids A (8) and B (9) [16], were identified by comparison of their observed NMR data with those reported in literatures.

All compounds were evaluated for their inhibitory effects on NO production induced by LPS in RAW264.7 macrophage cells. Compound 3 exhibited pronounced inhibition of NO production with an IC50 value of 13.44 ± 0.28 μmol·L-1, being comparable to the positive control, quercetin (IC50 17.0 ± 2.10 μmol·L-1). To investigate whether the inhibitory activities of compound 3 was generated from its cytotoxicity, the effects on LPS-induced RAW264.7 macrophage cells viability were measured using the MTT method. The result showed that compound 3 (up to 80 μmol·L-1) did not show any significant cytotoxicity with LPS treatment for 24 h.

In conclusion, a new diterpene with a 5/6/6/4 carbon ring system was isolated from Jatropha podagrica. This type of diterpenoid is rare in nature and only 16 derivatives have been reported so far [13, 17-20]. Compound 1 represents the first example of this compound class from genus Jatropha. Biosynthetically, the 5/6/6/4 carbon ring system might derive from the intramolecular cyclization of the macrocyclic deterpenes skeleton such as the co-isolated lathyranes 4 and 5 [13]. Moreover, compound 3 showed pronounced anti-inflammatory activities against nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells, which expended the pharmaceutical usage of this plant.

Experimental General Experimental Procedures

The X-ray data were collected using an Agilent Xcalibur Nove X-ray diffractometer. Melting points were measured on an X-4 melting instrument and were uncorrected. Optical rotations were measured on a Rudolph Autopol I automatic polarimeter, UV spectra on a Shimadzu UV-2450 spectrophotometer, and IR spectra on Bruker Tensor 37 infrared spectrophotometers. 1D and 2D NMR spectra were measured on Bruker AM-400 spectrometers at 25 ℃. ESIMS was measured on a Finnigan LCQ Decainstrument, and HR-ESIMS was performed on a Waters Micromass Q-TOF spectrometer. Column chromatography was performed on silica gel (300-400 mesh, Qingdao Haiyang Chemical Co., Ltd.), reversed-phase C18 (RP-C18) (12 nm, S-50 μm, YMC Co., Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and MCI gel (CHP20P, 75-150 μm, Mitsubishi Chemical Industries Ltd.). A Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector was used for HPLC. An YMC-pack ODS-A column (250 mm × 10 mm, S-5 μm, 12 nm) and a Phenomenex Lux cellulose-2 chiral column (10 mm × 250 mm, 5 μm) were used for semi-preparative HPLC separation. All solvents used were of analytical grade (Guangzhou Chemical Reagents Company, Ltd.).

Plant material

The stem of Jatropha podagrica were collected in the Xishuangbanna city, Yunnan Province, China, in May 2016, and were authenticated by Associate Professor XU You-Kai of Xishuangbanna Tropical Botanical Garden. A voucher specimen (accession number: FDS-201605) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

Extraction and isolation

The air-dried powder of the stems of J. podagrica (10 kg) was extracted with 95% EtOH (3 × 10 L) at room temperature to give a crude extract (600 g). The extract was suspended in H2O (2 L) and successively partitioned with petroleum ether (PE, 3 × 2 L), and EtOAc (3 × 2 L) to yield two corresponding portions. The EtOAc extract (100 g) was subjected to MCI gel column eluted with MeOH-H2O (5 : 5 → 10 : 0) to afford Frs. Ⅰ-Ⅳ. Fr. Ⅱ was subjected to silica gel column (PE/EtOAc, 10 : 1 → 0 : 1) to afford Frs. IIA−IID. Fr. IID was loaded onto a Sephadex LH-20 column and eluted with MeOH to give Fr. IID1-Fr. IID2, then Fr. IID2 was subjected to silica gel column (PE/CH2Cl2, 1 : 1 → 0 : 1) to afford 4 (8 mg) and 5 (10 mg). Separation of the Fr. Ⅲ (14 g) was subjected to silica gel column (PE-CH2Cl2, 10 : 1 → 0 : 1) to get Fr. ⅢA and ⅢB. Fr. ⅢA was subjected to silica gel column (PE-EtOAc, 20 : 1 → 1 : 1) to afford Fr. ⅢA1 and ⅢA2. Fr. ⅢA1 was purified on HPLC with a Phenomenex Lux chiral column (CH3CN- H2O, 4.5 : 5.5, 3 mL·min-1) to obtain 1 (2 mg, tR 11 min). Fr. ⅢB was loaded onto a Sephadex LH-20 column and eluted with MeOH to give Fr. ⅢB1-Fr. ⅢB3. Fr. ⅢB1 (200 mg) was subjected to silica gel column (PE-EtOAc, 20 : 1 → 1 : 1) to afford Fr. ⅢB1a−Fr. ⅢB1c, then Fr. ⅢB1a was purified on a HPLC system equipped with a YMC column (MeOH-H2O, 8 : 2, 3 mL·min-1) to give 2 (28 mg, tR 13 min) and 3 (35 mg, tR 17 min). Fr. ⅢB1b was purified on HPLC (MeOH-H2O, 7.5 : 2.5, 3 mL·min-1) to give 6 (18 mg, tR 9 min) and 7 (16 mg, tR 12 min). Fr. Ⅳ was separated by column chromatography on reversed-phase silica gel C18 eluted with a MeOH-H2O gradient (5 : 5 → 10 : 0) to give Fr. IVA-Fr. IVD, and then Fr. IVB was loaded onto a Sephadex LH-20 column and eluted with MeOH to afford Fr. IVB1 and Fr. IVB2, and then Fr. IVB2 was subjected to further purification by HPLC with a Phenomenex Lux chiral column (MeOH-H2O, 9.2 : 0.5, 3 mL·min-1) to give 8 (8 mg, tR 18 min) and 9 (10 mg, tR 22 min). The purity of compounds 1-9 was greater than 95% as determined by 1H NMR spectrum.

Jatrogricaine A (1)

Colorless crystal, mp 205-210 ℃; [α]20D +36 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 207 (3.67) nm, IR (KBr) νmax 3553, 3408, 2948, 2924, 2864, 1702, 1671, 1460, 1393, 1373, 1320, 1289, 1245, 1228 cm-1; 1H and 13C NMR data, see Table 1; HR-ESIMS m/z 355.1880 [M + Na]+ (Calcd. for C20H28O4Na, 355.1880).

Table 1 1H (500 MHz) and 13C (125 MHz) NMR data of 1 in CDCl3
X-ray crystallographic analysis of 1

C20H28O4, M = 332.44, monoclinic, space group P21 (no. 4), a = 9.9044 (1) Å, b = 7.4188 (1) Å, c = 12.5208 (2) Å, β = 99.585 (1), V = 907.17 (2) Å3, Z = 2, T = 294.7 (6) K, μ (Cu Kα) = 0.669 mm-1, Dc = 1.2170 g·cm-3, 16576 reflections measured (7.16° ≤ 2θ ≤ 144.12°), 3521 unique (Rint = 0.0257, Rsigma = 0.0166) which were used in all calculations. The final R1 was 0.0352 (I > = 2σ (I)) and wR2 was 0.0937. Flack parameter = -0.14 (13). Crystallographic data for the structure of 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CDCC 1589314).

Cell culture

The RAW264.7 mouse macrophage cell line was purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China), and was cultured in Dulbecco's modified Eagle medium (DMEM, Gibco Invitrogen Corp., Carlsbad, CA, USA) which was supplemented with 10% FBS, 100 Units/mL penicillin and 100 μg·mL-1 streptomycin. The cells were placed at 37 ℃ in a humidified incubator containing 5% CO2.

MTT assay

The cytotoxicity of the isolated compounds toward RAW264.7 cells was determined by MTT assay. RAW264.7 cells were planted in 96-well plates (5 × 103/well) for 24 h. Then they were treated with test samples which dissolved in DMSO and diluted in 100 μL DMEM making the final drug concentration 50 μmol·L-1 and 1% DMSO. 1% DMSO served as solvent control. Wells without cells contain only 100 μL DMEM were served as blank control. 24 h later, 20 μL solution MTT was added in each well. After incubation for 4 h, the medium was removed and 100 μL DMSO was added in each well, then the absorbance (A) was detected at 490 nm using a microplate reader. The inhibition of cell growth was calculated according to the following formula: % Inhibition = [1-(Asample-Ablank) / (Asolvent-Ablank)] × 100.

Inhibitory activity toward NO

NO release was assessed by a colorimetric assay based on a diazotization reaction using the Griess reagent system. RAW264.7 cells were planted in 96-well plates (5 × 103/well) for 24 h and then pre-incubated with different concentrations of compounds for 1 h before stimulation with LPS (1.0 μg·mL-1) for 24 h. The NO concentration in culture medium was determined by Griess reagent kit, then the absorbance (A) was measured at 540 nm using a microplate reader. The inhibition of NO release was calculated according to the following formula: Inhibition (%) = [1-(Asample-Ablank) / (Amodel-Ablank)] × 100. The experiments were performed in triplicates, and the data were presented as the mean ± SD. Quercetin was used as a positive control.

References
[1]
Editorial Committee of Chinese Flora. Flora of China[M]. Beijing: Science Press, 1996: 147.
[2]
Devappa RK, Makkar HPS, Becker K. Jatropha diterpenes: a review[J]. J Am Oil Chem Soc, 2011, 88(3): 301-322. DOI:10.1007/s11746-010-1720-9
[3]
Wang XC, Zheng ZP, Gan XW, et al. Jatrophalactam, a novel diterpenoid lactam isolated from Jatropha curcas[J]. Org Lett, 2009, 11(23): 5522-5524. DOI:10.1021/ol902349f
[4]
Chianese G, Fattorusso E, Aiyelaagbe OO, et al. Spirocurcasone, a diterpenoid with a novel carbon skeleton from Jatropha curcas[J]. Org Lett, 2011, 13(2): 316-319. DOI:10.1021/ol102802u
[5]
Liu JQ, Yang YF, Li XY, et al. Cytotoxicity of naturally occurring rhamnofolane diterpenes from Jatropha curcas[J]. Phytochemistry, 2013, 96(12): 265-272.
[6]
Aiyelaagbe OO, Adesogan K, Ekundayo O, et al. Antibacterial diterpenoids from Jatropha podagrica Hook[J]. Phytochemistry, 2007, 68(19): 2420-2425. DOI:10.1016/j.phytochem.2007.05.021
[7]
Ojewole JA, Odebiyi OO. Neuromuscular and cardiovascular actions of tetramethylpyrazine from the stem of Jatropha podagrica[J]. Planta Med, 1980, 38(4): 332-338. DOI:10.1055/s-2008-1074885
[8]
Ojewole JA, Odebiyi OO. Mechanism of the hypotensive effect of tetramethylpyrazine, an amide alkaloid from the stem of Jatropha podagrica[J]. Planta Med, 1981, 41(3): 281-287. DOI:10.1055/s-2007-971715
[9]
Liu WW, Zhang Y, Yuan CM, et al. Japodagricanones A and B, novel diterpenoids from Jatropha podagrica[J]. Fitoterapia, 2014, 98: 156-159. DOI:10.1016/j.fitote.2014.07.021
[10]
Bao JM, Su ZY, Lou LL, et al. Jatrocurcadiones A and B: two novel diterpenoids with an unusual 10, 11-seco-premyrsinane skeleton from Jatropha curcas[J]. RSC Adv, 2015, 5(77): 62921-62925. DOI:10.1039/C5RA11380F
[11]
Zhu JY, Lou LL, Guo YQ, et al. Natural thioredoxin reductase inhibitors from Jatropha integerrima[J]. RSC Adv, 2015, 5(58): 47235-47243. DOI:10.1039/C5RA07274C
[12]
Zhu JY, Wang RM, Lou LL, et al. Jatrophane diterpenoids as modulators of p-glycoprotein-dependent multidrug resistance (MDR): advances of structure-activity relationships and discovery of promising MDR reversal agents[J]. J Med Chem, 2016, 59(13): 6353-6369. DOI:10.1021/acs.jmedchem.6b00605
[13]
Remy S, Olivon F, Desrat S, et al. Structurally diverse diterpenoids from sandwithia guyanensis[J]. J Nat Prod, 2018, 81(4): 901-912. DOI:10.1021/acs.jnatprod.7b01025
[14]
Schmeda-Hirschmann G, Tsichritzis F, Jakupovic J. Diterpenes and a lignan from Jatropha grossidentata[J]. Phytochemistry, 1992, 31(5): 1731-1735. DOI:10.1016/0031-9422(92)83137-N
[15]
Liu JQ, Yang YF, Xia JJ, et al. Cytotoxic diterpenoids from Jatropha curcas cv. nigroviensrugosus CY Yang roots[J]. Phytochemistry, 2015, 117: 462-468. DOI:10.1016/j.phytochem.2015.07.002
[16]
Yang DS, Zhang YL, Peng WB, et al. Jatropholane-type diterpenes from Euphorbia sikkimensis[J]. J Nat Prod, 2013, 76(2): 265-269. DOI:10.1021/np300799n
[17]
Shi JG, Jia ZJ, Yang L. Diterpenoids from Euphorbia micractina[J]. Phytochemistry, 1992, 32(1): 208-210. DOI:10.1016/0031-9422(92)80136-3
[18]
Shi JG, Jia ZJ. Diterpenoids from Euphorbia micractina[J]. Phytochemistry, 1995, 38(6): 1445-1447. DOI:10.1016/0031-9422(94)00587-J
[19]
Vasas A, Hohman J, Forgo P, et al. New tri- and tetracyclic diterpenes from Euphorbia villosa[J]. Tetrahedron, 2004, 60(23): 5025-5030. DOI:10.1016/j.tet.2004.04.028
[20]
Tian Y, Xu WD, Zhu CG, et al. Diterpenoids with diverse skeletons from the roots of Euphorbia micractina[J]. J Nat Prod, 2013, 76(6): 1039-1046. DOI:10.1021/np400029d