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Pentacyclic Triterpenes in Euphorbia microsciadia with Their T-cell Proliferation Activity.

The ethyl acetate partition of dried methanolic extract of aerial parts of Euphorbia microsciadia (Euphorbiaceae) afforded three pentacyclic triterpenes, betulinic acid (1) from lupane type, oleanolic acid (2) from oleane type and ursolic acid (3) from ursane type triterpenes that are reported for the first time in this plant. These three compounds were structurally compared through their mass fragmentation pattern, nuclear magnetic resonance (NMR) data and their biologic immunomodulatory effects. The structures of the isolated compounds were elucidated by 13C- and 1H-NMR as well as 2D-NMR, IR and by the aid of mass fragmentation pattern and comparing with the literature. After running T-Cell proliferation assay, oleanolic acid stimulated proliferation of T-Cells at lower concentration 0.5 µg/mL, while betulinic acid and ursolic acid showed inhibitory activity against T-Cell proliferation with IC50- value > 50 µg/mL and 3.01 ± 0.47 µg/mL, respectively.


Introduction
Euphorbiaceae is one of the largest families of the phylum Anthophyta. In this family, the largest genus is Euphorbia which comprises well over 2000 species in tropical and temperate zones of Asia and other parts of the world. In Iran, 70 species are reported out of which 17 are endemic (1). In traditional medicine, Euphorbia was used as the treatment of intestinal parasites, gonorrhea, wart cures and also in the treatment of skin diseases (2). However, multidisciplinary biological screening tests carried out in recent years have shown that some of them were useful as anti-tumors, pesticides and antiviruses.
In this research, three triterpenes of three different common pentacyclic classes were isolated from the aerial parts of Euphorbia microsciadia (Euphorbiaceae) which is a perennial plant that grows in some parts of Iran (3); there has been efforts to elucidate them through Nuclear magnetic resonance (NMR) and other spectroscopic methods, especially the mass fragmentation pattern.

General
The 1 H-NMR spectra were recorded on a Bruker Avance AV 300 but 13 C-NMR and 2D-NMR spectra were recorded on a Bruker Avance AV 600 NMR instrument, using CDCl3 as solvent. The IR spectra were recorded on a JASCO 302-A spectrophotometer and the mass spectra (EI and HREI-MS) were measured in an electron impact mode on Varian MAT 112 or MAT 312 spectrometers. Fast atom bombardments (FAB) MS were measured on Jeol HX110 mass spectrometer.

Extraction and isolation
The dried plant (4.5 Kg) was extracted with by three times and the resulting extract was concentrated to a 400 g dark green gum. This gum was partitioned into n-heptane and aqueous MeOH (80%). The defatted MeOH extract was evaporated and dissolved in H 2 O. The aqueous extract was partitioned into H2O and ethyl acetate (EtOAc). Thereafter, EtOAc part was subjected on RP-18 column chromatography eluting with aqueous methanol 30%, 60% and 100% respectively. The fraction obtained on elution with H2O : MeOH (40 : 60) transferred on separated from the rich chlorophyll content and pigments by size exclusion chromatography on / MeOH (1 : 2). Finally, fractions which showed Jaigel-2H and 1H columns with chloroform as the mobile phase to yield three pure compounds (1-3) that were crystallized in DCM : MEOH (1 : 3).

T-Cell proliferation assay
Fresh venous blood from a normal healthy volunteer was collected in heparinized container and mixed with equal volume of RPMI-1640 (Mediatech Inc., Herndon, VA, U.S.A.), then layered onto lymphocyte separation medium mononuclear cell buffy coat was removed and cells were washed with incomplete RPMI-1640 peripheral blood mononuclear cells (PBMNCs) were resuspended in supplemented RPMI-1640 at 2.5×10 6 serum (FBS) from PAA laboratories GmbH (Pasching, Austria). In a 96-well round-bottomed 2 in air for 72 h. Further incubation for 18 h after the addition of thymidine [ 3 H] (Amersham, Buckinghamshire, UK) was done and cells were harvested using cell harvester(Inotech Dottikon, Switzerland) and the incorporation was measured by a liquid Fullerton, CA, USA) (4).

Statistical analysis
The IC 50 -values were calculated using an Excel based program and reported as mean ± was attributed to p-values (p < 0.05) and the probability values obtained by the student t-test between the sample and control data.

Results and Discussion
Compound 1 (6). 13 C-NMR (BB and DEPT) spectra showed thirty carbons comprised of seven methyls, eleven methylenes , six methines and six quaternary carbons. Based on above observations, the' spectral data of compound 1 was similar to those previously reported of betulinic acid (5, 6), except for the assignment authors decided to assign it again by Double-Quantum Filtered Correlation Spectroscopy  17 (dt, H 22b ). Fragment S 1 , due to its attachment to hydroxyl group, proposed to be C 1 -C 2 -C 3, HMBCs of H 5 with C 1 S 2 as C 5 -C 6 H 2.36 (H 19 ) with c 150.62 (C 20 ), assigned substructure S 3 as C 11 -C 12 -C 13 -C 18 -C 19 -C 21 -C 22 c = 150.6 as C 20 . The position of six singlet methyls, Me23, Me24, Me25, M26, Me 27 and Me30 bonded to quaternary carbons C 4 , C 8 , C 10 , C 14 and C 20 were determined by their

Conclusion
A large number of pentacyclic triterpenes properties. Among these are ursolic acid and the lupane-type triterpenes like betulinic acid. Both of these compounds have been tested in a number of in-vitro and in-vivo model systems (9-13). Betulinic acid possess moderate inhibitory activity at relatively high concentrations on in-vitro was in agreement of other published data (12). Ursolic acid was found to potently inhibit T-cell proliferations while the oleanolic acid showed dose-relative stimulatory effect on lymphocyte proliferation assay agreed with another study by Anamika Khajuria (9) which showed dose dependent immunostimulatory effect on invivo immune functions in mice. Comparing the results, oleanolic acid and ursolic acid with the same structure differ only in E ring, showing different activities (one inhibitory indicating that the E ring (and not A) could be responsible for these changes. On the other hand, ursolic acid was found to potently inhibit T-cell proliferations more than betulinic acid indicating that the activity of six-member E ring the combination of E ring size as well as C-19, C-20 and C-28 positions could be responsible for the differences in biological effects in pentacyclic triterpenes analogues. Hence, positions like C-28 amino-derivatives, changing the C-20 alkene in betulinic acid derivatives, changing the nucleophilicity and the strength of hydrogen bonding capability and/or acidity at position C-28 are suggested to improve their immunomodulatory activities.