New tigliane-type diterpenoids from Euphorbia aellenii Rech. f. with immunomodulatory activity.

The cytotoxic chloroform fraction of Euphorbia aellenii Rech. F. (Euphorbiaceae) afforded two new phorbol diterpenoids: 4-deoxy-4α-phorbol-12-(2,3-dimethyl) butyrate-13-isobutyrate and 17-hydroxy-4-deoxy-4α-phorbol-12-(2,3-dimethyl) butyrate-13-isobutyrate. Their structures were elucidated by NMR and other spectroscopic methods. The immunomodulating potentials of the isolated compounds were tested using standard proliferation and chemiluminescence assays. Compound 2 showed moderate inhibitory activity against both T-cell proliferation and reactive oxygen species (ROS) production in whole blood with IC50 of 14.0 ± 0.57 and 44.1 ± 3.8 μg/ml, respectively, while compound 1 was relatively inactive with IC50 >50 μg/mL for T-cell proliferation, and >100 μg/mL for ROS.

INTRODUCTION

Many of the published studies on Euphorbia family have highlighted their immunomodulating, anti-tumor, and anti-HIV properties, more probably related to the presence of certain types of polycyclic diterpenes (1). Diterpenes constitute a group of C20 compounds arising from geranyl geranyl pyrophosphate. Cyclization of the diterpenes leads to the formation of polycyclic structures by intramolecular nucleophilic substitution i.e. jatrophane, lathyrane, mirisinane and tigliane diterpenes in Euphorbia family (2). Among them the tigliane nucleus is the carbon frame-work of phorbol whose derivatives occur widely in Euphorbiaceae and are renowned for their tumor promoting and irritant activities (2–3).

Recently, lathyrane diterpenes and myrosinol type skeletons related to lathyranes have been isolated from this plant (4–5). In the present study, two new tigliane type derivatives were isolated and their structures were elucidated by NMR and other spectroscopic methods. Immunomodulating potentials of the isolated compounds were tested by chem-iluminescence assay using neutrophils of human whole blood.

MATERIALS AND METHODS

Materials

Calcium chloride and magnesium sulphate purchased from Sigma-aldrich (USA), luminol (3-aminophthalhydrazide) from Research Organics (USA), serum opsonized zymosan (Saccharomyces cerevisiae origin) from Fluka (Switzerland) and thymidine [3H] from Amersham (UK). All solvents used were of analytical grade and purchased from Merck (Germany).

NMR and IR spectroscopy

NMR spectra were recorded on a Bruker Avance AV 300 and AV 600 NMR instrument (Switzerland) using CDCl3 as solvent. The IR spectra were recorded on a JASCO 302-A spectrophotometer (Japan), EI-MS spectra were measured in an electron impact mode on Varian MAT 112 or MAT 312 spectrometers.

Chromatographic condition

Recycling preparative HPLC was carried out on an LC-908 (Hitachi Co., Japan) equipped with UV and RI detectors using a YMC-Pack-Sil column (250 × 20 mm i.d.). Flash chroma-tography was performed on LiChroprep® Si 60 (25-40 μm), or silica gel 63-200 μm, and size exclusion chromatography on Sephadex® LH-20 (Sigma-Aldrich). HPTLC was conducted on precoated silica gel GF-254 plates (20 × 20 cm, 0.5 mm thick) (Merck, Germany) and visualization of the plates was achieved at 254 /366 nm.

Plant materials

The aerial flowering parts of the Euphorbia aellenii Rech. f. (Euphorbiaceae) were collected in August 2007 from populations growing in Galil-e-Shirvan (Alt. 1600 m), Northern Khorasan province (Iran) and identified by Mrs. Yasamin Naseh, plant taxonomist (Department of Botany, Herbaceous Sciences Research Center at the Ferdowsi University of Mashhad). A voucher specimen (no. 2024) of the plant has been deposited in the herbarium of the Pharmacognosy Department, School of Pharmacy and Pharmaceutical sciences at the Isfahan University of Medical Sciences (Iran).

Extraction and isolation

Air-dried plant was ground to fine powder (7 kg) and macerated for 4 days with MeOH (20 L × 3) at room temperature. Filtration and in vacuo evaporation resulted in a green gum (500 g) which was partitioned between methanol and n-hexane. The defatted extract was concentrated, dissolved in water, and successively extracted with chloroform, ethyl acetate and n-butanol, respectively. The obtained fractions were compared in vitro for their cytotoxic activities against brine-shrimp eggs (6). Consequently, the chloroform fraction (240 g) with potential of cytotoxic activity was subjected to column chromatography over normal silica gel, using Hexane/CHCl3 mixtures of increasing polarity up to 100%. Hexane/CHCl3 (20:80) was further purified using gradient mixtures of Hexane/EtOAc (0→50) on silica. Next, the diethyl ether soluble part of Hexane/EtOAc (70:30) fraction was purified on sephadex LH-20 (DCM/MeOH, 1:2) followed by RP-18 CC (MeOH/H2O, 70:30) to remove chlorophylls and pigments. Finally, fractions containing diterpenes (inferred from 1H-NMR spectra) were subjected to recycling HPLC (Hexane/ EtOAc, 70:30) to obtain compounds 1 (11.0 mg) and 2 (8.0 mg).

Phagocyte chemiluminescence assay

In stimulated polymorphonuclear cells (PMNs), inhibition of chemiluminescence may be mediated by three main mechanisms including cell death, scavenging of ROS and inhibition of enzymes involved in the signal transduction pathways of the ROS generation process. In this assay, formation of the reactive oxidants in whole blood during the oxidative burst was measured by the luminol-enhanced chemiluminescence assay procedure (78). In brief, three concentrations (1, 10 and 100 μg/mL) of each compound were prepared in 25 μL of Hank's Buffered Salt Solution (HBSS++) in 96 well flat bottomed plates for a final incubation volume of 100 μL. Then 25 μL of whole blood diluted 1:50 in suspension of HBSS++ with calcium chloride and magnesium sulphate was added. Positive control, negative control and blank wells were included in the assay. Cells and compounds were incubated for 30 min at 37° C. 25 μL luminol (3-aminophthalhydrazide) was then added into each well and 25 μL serum opsonized zymosan (Saccharomyces cerevisiae origin) was added except for negative and blank wells. The phagocytosis kinetic was monitored with luminometer (Labsystems Luminoskan, Finland) for 50 min in the repeated scan mode. Peak and total integral chemiluminescence readings were expressed in terms of relative light unit (RLU).

T-Cell proliferation assay

Peripheral human blood lymphocytes were incubated with different concentrations of the test compounds (0.5, 5, and 50 μg/mL in duplicates) in supplemented RPMI-1640 along with phytohemagglutinin (PHA) at 37 °C in CO2 environment for 72 h. Further incubation for 18 h after the addition of thymidine [3H] was done and cells were harvested using a cell harvester (Inotech Dottikon, Switzerland). Finally, proliferation level was determined by the radioactivity count as CPM reading record-ed from the Beta-scintillation counter (5).

Statistical analysis

All samples were presented as mean ± SD for three measurements. One-way ANOVA was used to calculate P <0.05 for each compound against the control (+ve) and the IC50 values were calculated using Excel 2007.

RESULTS

Chemistry

Two new compounds (1–2) were obtained with the following NMR and spectroscopic properties and assigned aided by the 1H- 1H COSY, and HMBC experiments (Fig. 1).

Fig. 1

Key DQF-COSY (in bold) and (H→C) HMBC correlations observed in tigliane-type diterpenes from Euphorbia aellenii

Compound 1

Colourless oil, UV (CHCl3) λmax 239 nm. IR (KBr+CHCl3) υmax 3733, 3610, 2873, 1714, 1645, 1517, 1456, 1394, 1204, 1160, 1076, 1029 cm-1; 1H NMR (CDCl3, 300 MHz, J in Hz): δ 7.03 (1H, bs, H-1), 5.45 (1H, d, J=10.02, H-12), 5.09 (1H, bs, H-7), 4.00 (1H, d, J=12.3, H- 20b), 3.89 (1H, d, J=12.3 H-20a), 3.46 (1H, bd, H-10), 3.42 (1H, bd, H-5a), 2.75 (1H, m, H-4), 2.51 (1H, overlapped, iBu-2“), 2.43 (1H, dd, J=15.3, 5.1, H-5b), 2.20 (1H, dq, J=7.2, 7.2, diMeBu-2’), 1.94 (1H, overlapped, H-8), 1.88 (1H, overlapped, diMeBu-3’), 1.76 (3H, s, H-19), 1.67 (1H, dq, J=10.8, 6.3, H-11), 1.18 (3H, s, H-17), 1.15 (3H, d, J=7.2, IBu-3”), 1.14 (3H, s, H-16), 1.13 (3H, d, J=7.2, diMeBu-5’), 1.11 (3H, d, J=7.2, iBu-4”), 1.07 (3H, d, J=6.3, H-18), 0.96 (3H, d, J=7.2, diMeBu-4’), 0.92 (3H, d, J=7.2, diMeBu-6’), 0.75 (1H, d, J=5.1, H-14); 13C NMR (CDCl3, 125 MHz): δ 213.1 (C-3), 178.9 (iBu-C-1”), 175.6 (diMeBu-C-1’), 156.1 (C-1), 143.2 (C-2), 137.1 (C-6), 126.4 (C-7), 78.0 (C-9), 74.8 (C-12), 69.3 (C-20), 64.8 (C-13), 49.6 (C-4), 47.3 (diMeBu-C-2’), 47.3 (C-10), 43.1 (C-11), 40.7 (C-8), 37.1 (C-14), 34.3 (iBu-C-2”), 31.1 (diMeBu-C-3’), 25.2 (C-15), 25.2 (C-5), 24.1 (C-16), 20.8 (diMeBu-C-6’), 19.3 (diMeBu-C-4’), 18.5 (iBu-C-3”), 18.5 (iBu-C-4”), 16.4 (C-17), 14.5 (diMeBu-C-5’), 11.8 (C-18), 10.4 (C-19); HRESI-MS, Positive mode: m/z 517.3207 (calc. for C30H44O7 +H+, 517.3159), 383 , 354, 313, 295.

Compound 2

Colourless oil, UV (CHCl3) λmax (log ε): 232 (3.95), 280 (3.68) nm. IR (KBr+CHCl3) υmax 3799, 3733 ,3610,3970, 2873,1725,1714, 1680, 1645,1517,1456, 1394,1250, 1204,1160, 1076,1029, 667,408 cm-1; 1H NMR (CDCl3 + CD3OD, 300 MHz, J in Hz): δ 7.00 (1H, bs, H-1), 5.45 (1H, d, J=10.5, H-12), 5.06 (1H, bs, H-7 , 3.90 (1H, d, J=13.1, H- 20b), 3.82 (1H, d, J=13.1, H-20a), 3.57 (1H, br-s , H-17), 3.43 (1H, bd, H-10), 3.33 (1H, overlapped, H-5a), 2.75 (1H, m, H-4), 2.45 (1H, m, iBu-2”), 2.33 (1H, dd, J=15.3, 4.8, H-5b), 2.17 (1H, dq, J=6.9, 6.99, diMeBu-2’), 1.89 (1H, bd, H-8), 1.84 (1H, m, diMeBu-3’), 1.71 (3H, s, H-19), 1.64 (1H, dq, J=10.8, 6.3, H-11), 1.15 (3H, s, H-16), 1.15 (3H, d, J=7.2, diMeBu-6’, 1.10 (3H, overlapped, diMeBu-5’), 1.08 (3H, d, J=6.9, IBu-3”), 1.06 (3H, d, J=6.9, iBu-4”), 1.01 (3H, d, J=6.3, H-18), 0.93 (3H, d, J=6.9, diMeBu-4’), 0.72 (1H, d, J=4.8, H-14); 13C NMR (CDCl3+CD3OD, 125 MHz): δ 213.0 (C-3), 179.1 (iBu-C-1”), 175.1 (diMeBu-C-1’), 156.5 (C-1), 143.3 (C-2), 136.0 (C-6), 125.2 (C-7), 78.0 (C-9), 74.9 (C-12), 70.4 (C-17), 68.8 (C-20), 65.7 (C-13), 49.8 (C-4), 47.3 (diMeBu-C-2’), 47.2 (C-10), 42.9 (C-11), 40.7 (C-8), 37.0 (C-14), 34.2 (iBu-C-2”), 31.1 (diMeBu-C-3’), 25.3 (C-15), 25.3 (C-5), 24.0 C-16), 20.7 (diMeBu-C-6’) 19.2 (diMeBu-C-4’), 18.4 (iBu-C-3”), 18.4 (iBu-C-4”), 14.5 (diMeBu-C-5’), 11.7 (C-18), 10.3 (C-19); HRESI-MS, Positive mode: m/z 533.3165 (calc. for C30H44O8 +H +, 533.3109), 515, 417, 399, 354, 329, 301, 217.

Lymphocyte proliferation assay

The anti-proliferation effect of the test compounds was determined by measuring the PHA-induced T-cell proliferation by determining radioactive thymidine incorporation. Compound 2 showed inhibitory activity against lymphocyte proliferation with IC50 of 13.27 ± 0.18 μg/mL whereas compound 1 was reactively inactive (IC50 >50 μg/mL).

Chemiluminescence assay

The concentration effects of the tested compounds on human whole blood employing luminol and zymosan for the intracellular oxidative burst studies are presented in Fig. 2). The inhibitory effect of compound 1 at the concentrations of 100 (P <0.001) and 10 μg/ml (P <0.01) was significantly greater than positive control. This effect, however, did not reach to a significant level at concentration of 1 μg/ml. The inhibitory effect of compound 2 at the concentration of 100 μg/ml was markedly larger than that of the control (p<0.0001), but not at concentrations of 10 and 1 μg/ml.

Fig. 2

Effects of compounds 1 and 2 on the neutrophils oxidative burst. The luminol dependent chemiluminscence induced by zymosan in the presence of compounds 1 and 2 at three concentrations are compared to those of the positive control (+ve). Data are presented as means ± SD for three measurements. One-way ANOVA was used to analyze the differences between the inhibitory effects of each compound and positive control. Stars show statistically significant differences between the test and control. *P<0.01, **P<0.001, ***P<0.0001 (ANOVA).

Weak inhibition activity was observed for compound 1 with the IC50 >100 μg/ml, while moderate inhibitory activity with IC50 equal to 44.1 ± 3.84 μg/ml was found for compound 2 , which may be due to the inhibition of PMNs proliferation.

DISCUSSION

Compound 1 obtained as colourless oily mass was assigned the molecular formula of C30H44O7 on the basis of positive HRESI-MS, m/z 517.3207 (calc. for C30H44O7 +H +, 517.3159), in accordance with number of carbons and hydrogens counted in NMR data. The IR spectrum confirmed the presence of carbonyls (1675-1750 cm-1), C-O (1020-1250 cm-1), double bond absorption (1645, 1517 cm-1) and free hydroxyls (3733, 3610 cm -1). According to the nine degrees of unsaturation derived from molecular formula, 13C-NMR and DEPT spectral data, one ketone group, two ester carbonyls, two double bonds, four rings have been deduced in the molecule. In the 1H-NMR spectrum, four methyl groups 5r 1.07 d (6.3), 1.14 s, 1.18 s and 1.76, two geminal oxymethylene protons δH 4.00 (d, J=12.3 Hz, H-20b) and 3.89 (d, 12.3 Hz, H-20a), two geminal hydrogens δH 2.43 (dd, J= 15.3, 5.1 Hz, H-5a) and 3.42 (br-d, H-5b) together with two olefinic protons δH 7.03 (br-s, H-l) and 5.09 (br-s, H-7) as well as the methine doublets at ca δH =5.4 (J ~ 9-10 Hz) and at ca δH =1 (J~5 Hz) attributed to H-l2 (5.45, d, J=10.2 Hz) and H-14 (0.74, d, J=5.1 Hz), respectively showed typical signals of phorbol esters (8-10). HMBC correlation of δH 7.03 of an olefinic group with ketone carbonyl δc 213.1 along with IR absorption (1675 cm-1) indicated one α, β-unsaturated ketone system. Moreover, signals of the cyclopropane moiety, C-13 (δc =64.8) and C-15 (δc =25.3) together with HMBC correlations with two singlet methyl groups at δH 1.14 and 1.18 with quaternary carbon δc 25.2 (C-15) confirmed the presence of these two geminal unfunctionalized methyl groups on cyclopropane ring (8, 11). The 1 H-1H -COSY correlations confirmed the following protons : CH3-CH-CH(CH3)-CH3 [δH 1.13 d (7.2), 2.20 dq (7.2; 7.2), 1.88 m) and 0.92 d (7.2)] to be in one spin system and presence of ion peak, m/z 400 in MS spectrum (M-116), as well as HMBC of H-12 (δH 5.45) and H-2’ (δH 2.20) with ester carbonyl carbon (δc 175.5) confirmed this fragment as 2’, 3’-dimethyl-butanoate moiety attached to C-12 (12). Likewise, 13C- and 1H-NMR data δ 178.9, 34.3 (2.51, m), 18.5 (1.15, d, J=7.2 Hz) and 18.5 (1.11, d, J=7.2 Hz) exhibited typical signals of isobutanoate group (8). Lack of HMBC correlations of isobutanoate group with any oxygenated carbons, suggested its position on a quaternary C-O (C-13) and it was confirmed by NOESY effects of Me-3” (δH 1.15) with H-14 (δH 0.74) denying its position on C-9 (8, 13). Therefore, in light of above observations, accordance to NMR data with literature and HMBC and DQF-COSY spectra (Fig. 1), compound 1 recognized as a 4α-deoxy tigliane, bearing one -CH2 OH group (δC 68.8) on C-6.

The stereochemistry of compound 1 was obtained by the analysis of NOESY spectra and J-coupling constants. According to the literature, all up to now have been discovered from plants, have shown β configuration for H-8 and α-orientation for C-9-OH and H-10 (1011). Therefore, with considering those as references, the NOE effects of H-10α with Me-18; Me-18 with H-12 andH-4 supported a position for these protons, in which the latter is confirmed by lack of NOE between H-4 and H-8β, as well. The large J-coupling constant (10.2 Hz) between H-12α and H-ll revealed their trans orientation and consequently the observed NOE of H-llβ with Me-17 disclosed that gem-dimethyl cyclopropane moiety were on the plane and the three-membered ring cis joined to seven-membered ring (Fig. 3). Therefore, based on above explanations, compound 1 was identified as 4-deoxy-4α-phorbol-12-(2,3 -dimethyl)butyrate-13 -isobutyrate .

Fig. 3

Key correlations observed in the NOESY spectrum of compound 1 from Euphorbia aellenii.

The molecular formula of compound 2 was assigned as C30H44O8 by the positive HRESI-MS, m/z 533.3165 (calc. for C30H44O8 +H +, 533.3109), according to the number of carbons and hydrogens counted in NMR data. IR spectrum showed a prominent peak of carbonyls (1680-1725 cm-1), olefinic group (1645 cm-1), C-O functions (1029-1250 cm-1) and hydroxyl groups (3610-3733 cm-1). The nine degree of unsaturation, 13C-NMR and DEPT spectral data supported the presence of three carbonyls (one ketonic and two esteric), two double bonds and consequently, four rings in the molecule. More observation in NMR data showed close similarity with that of compound 1, except for an additional free hydroxyl group on C-17 (δc 70.4) which proposed compound 2 to be 17-hydroxy-4-deoxy-4α-phorbol-12-(2,3-dimethyl) butyrate-13-isobutyrate diterpenoid.

Immunomodulating potential of the isolated compounds tested in standard proliferation and chemiluminescence assays showed moderate inhibitory activity against both T-cell prolifer-ation and ROS production in whole blood for compound 2 with IC50 of 14.0 ± 0.57 and 44.1 ± 3.8 μg/ml, respectively, while compound 1 was relatively inactive with IC50 >50 μg/mL for T-cell proliferation and >100 μg/ml for ROS. Similar studies on closely related phorbol derivatives for stimulation of human mononuclear cells have shown that 4β -deoxyphorbol esters stimulated cell proliferation in a dose-related manner, while 4α -deoxyphorbol esters had no effects which is in agreement with the results of the present study (15). In another study on the effects of various phorbol-based protein kinase C (PKC) activators which resulted in proliferation of T cells, 4β -phorbol 12,13-dibutyrate showed activity in a concentration-dependent manner, whereas the structurally related isomer 4α -phorbol 12,13-dibutyrate was inactive (16). Indeed, there is conflicting data as to whether 4-deoxy-phorbol esters are lymphocyte mitogens. This issue is important as phorbol esters are often regarded as proinflamatory promoters. In the current study and previous reports using related 4α -phorbol esters either no response or weak response to lymphocyte proliferation has been observed indicating that H-4 orientation is crucial to impart this effect.

CONCLUSION

Using size exclusion chromatography on Sephadex LH 20 and recycling HPLC with normal-phase column as powerful means for isolating phorbol esters, two new 4-deoxy-4α-phorbol esters were isolated from Euphorbia aellenii Rech. F., and their structures were elucidated by NMR and other spectroscopic methods. Immunomodulating potential of the isolated compounds was tested by phagocyte chemiluminescence and T-cell proliferation assays. These compounds showed moderate inhibitory activity on phagocytosis oxidative burst on polymorphoneutrophils (PMNs) in human whole blood and lymphocyte prolifera-taion. Therefore, E.aellenii could be a new source of theses chemo-type lead-compounds as starting material for semi-synthetic tiglians for discovering immunomodulating agents which are currently used to modulate the host natural defense and immune function in various conditions such as treatment of infections, organ rejection, rheumatoid arthritis and systemic lupus erythematous (14).