1. Background
Tripleurospermum disciforme (C. A. Mey.) Schultz-Bip. belongs to the Compositae (Asteraceae) family. Known as ‘‘Iranian Babouneh’’, it is a perennial herb that grows wildly to about 10 - 70 cm in height. The plant synonyms are Chrysanthemum disciforme (C. A. Mey.), Matricaria disciforme (C. A. Mey.) DC., Chamaemelum disciforme (C. A. Mey.) Vis. as well as Chamaemelum disciforme var. quadrilobum Boiss (1). It is believed that in Iranian traditional medicine (particularly in Hamedan and Mashhad provinces), decoction of T. disciforme is used for treatment of kidney stone (2), treatment of cough and febrifuge (3) and acting as anti-spasmodic and anti-inflammatory agent (4). According to the previous studies on various T. disciforme extracts and essential oils, several pharmacological and biological activities such as anti-oxidant (5, 6), anti-inflammatory (7), anti-ulcer (8), anti-bacterial (9, 10) and anti-fungal (11) have been reported. Meanwhile, recent phytochemical analysis of Tripleurospermum species extracts have revealed the presence of dioxaspirans (6), flavonoids (10), melatonins (12), triterpenes (13), acetylenes (14), and saponins (15). Herein, the isolation and structural identification of one taraxastane triterpene 1 and two lupane triterpenes 2 and 3 (Figure 1) are reported for the first time from the species mentioned. Among different kinds of studies evaluated in vitro, (1) taraxasterol; (2) lupeol; and (3) betulinic acid had cytotoxic effects on the various cancerous cell lines such as breast, cervix, colon, prostate, lung, ovary, skin, hematopoietic and lymphoid tissue (16-18).
Figure 1.
Chemical structures of isolated triterpenoids 1 - 3 from the aerial parts of T. disciforme
2. Objectives
The present study was designed to isolate and elucidate the natural terpenoids form the dichloromethane (DCM) extract of Tripleurospermum disciforme aerial parts (Compositae/Asteraceae). Moreover, not only the cytotoxic activity of the obtained compounds were assayed on two cancerous cell lines, AGS and WEHI-164, but the molecular duking of isolated compounds were also studied computationally by using AutoDock Vina.
3. Methods
3.1. Plant Material
The aerial parts of Tripleurospermum disciforme were collected in July 2014 from Taleghan (Tehran province) and were dried in the air. The plant was identified by Hamid Moazzeni (Traditional Medicine and Materia Medica Research Center), TMRC, Shahid Beheshti University of Medical Sciences, Tehran, Iran), and a voucher specimen (no. 3245) was deposited in the herbarium of the same institute.
3.2. Extraction and Isolation
The air-dried powdered aerial parts (200 g) of T. disciforme (C. A. Mey.) were extracted at room temperature with DCM by maceration method (3 × 2 L, rt for 24 h) and the combined extracts evaporated to give a dark green gummy residue (15 g). Five g of the extract was subjected on a silica gel (70 - 230 mesh) column chromatography (CC) and eluting with n-hexane:EtOAc:MeOH [100:0:0, 95:05:0, 90:10:0, 80:20:0, 70:30:0, 60:40:0, 50:50:0, 30:70:0, 10:90:0 and 0:80:20 (v/v), respectively] to yield ten fractions, A-J. Fraction E (277 mg) was re-chromatographed by Et2O:EtOAc in order of increasing polarities to yield 1 (43 mg, with Rf = 0.7 for Et2O:EtOAc, 5:5) and 2 (25 mg, with Rf = 0.6 for Et2O:EtOAc, 7:3). Compound 3 (19 mg, with Rf = 0.65 for CHCl3:EtOAc, 15:5) was purified from the fraction G (326 mg) by twice silica gel column chromatography. The first silica gel column (230 - 400 mesh) was prepared by stepwise gradient eluting with CHCl3:EtOAc, 10:0 - 8:2, and 7 subfractions were obtained. Consequently, the fifth subfraction loaded over second silica gel column (230 - 400 mesh) and isocratically eluted by CHCl3:MeOH, 98:2. The 1H-13C NMR along with mass spectroscopic data for (1) taraxasterol (19); (2) lupeol (20); and (3) betulinic acid (21) have previously been reported.
3.3. General Experimental Procedures
1H and 13C-NMR spectra (in CDCl3) were measured on a Bruker Avance TM 400 DRX spectrometer with TMS as an internal standard. Electron ionization mass spectra (EI-MS) were acquired using a Bruker Apex II mass spectrometer (Bruker, Bremen, Germany). Silica gel (70 - 230 and 230 - 400 mesh, Merck) were used for column chromatography. TLC was performed on Merck F254 silica gel plates (10 × 10 cm).
3.4. Cell Lines and Reagents
Two cell lines including AGS and WEHI-164; both from Pasteur Institute, Tehran, Iran, were grown in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, USA) containing 10% fetal bovine serum (FBS; GIBCO, USA) at 37ºC in a humidified atmosphere of 5% CO2.
3.5. MTT Cytotoxicity Assay
Cell viability was measured by MTT assay according to the manufacturer's instructions (Sigma-Aldrich, USA). Dendrosmal curcumin as an anti-proliferative compound was also employed as a positive control (22, 23). The relative cell viability was determined at 540 nm by a 96-well plate reader (Biorad-USA) and the concentration at which cell growth was inhibited by 50% (IC50) was determined by standard curve method (24, 25).
3.6. Computational Chemistry
3.6.1. Ligand preparation
The structures of isolated compounds were drawn by using ChemDraw Ultra (version 8.0 CambridgeSoft Corporation) and the 3D structures were optimized by Gaussian W90 software using semi empirical, PM3 level. The ligand structures were prepared by PyRx software and saved as pdbqt using autodock menu.
3.6.2. Receptors Preparation
The 3D X-ray structure of proteins Bcl-2, Bcl-xl, CDK2 and tubulin were obtained from RCSB database (PDB codes: 4aq3, 3spf, 2wlh and 4eb6, respectively). The presence of Vina plugin in PyRx software was used for the docking studies.
3.6.3. Docking
The grid boxes were constructed around the binding site of all proteins mentioned. The number of grid points in the three dimensions [npts] and coordinates by way of three dimensions [gridcenter] were in the following order;
Bcl-2 protein; [npts]: X: 40.63, Y: 34.76, Z: 39.13; [gridcenter]: X: -13.929, Y: 14.177, Z: -10.172.
Bcl-xl protein; [npts]: X: 42.81, Y: 36.67, Z: 37.65; [gridcenter]: X: 35.054, Y: 14.895, Z: -13.164.
CDK2 protein; [npts]: X: 52.49, Y: 40.53, Z: 63.72; [gridcenter]: X: 28.513, Y: 5.046, Z: 49.971.
Tubulin protein; [npts]: X: 17.07, Y: 15.67, Z: 17.78; [gridcenter]: X: 11.357, Y: 89.419, Z: 107.420.
The spacing and exhaustiveness ranges for all cases were determined 1.000 and 15, respectively. Hydrogen atoms and Gasteiger partial charges were added using AutoDock software (V. 4.2).
4. Results and Discussion
The DCM extract of T. disciforme aerial parts was fractioned by a normal phase-column chromatography (NP-CC) system, and subsequently three major triterpenoids were isolated. The triterpenoids exhibited violet spots on TLC plates by spraying Anisaldehyde-H2SO4 reagent followed by heating. The structure elucidations of (1) taraxasterol (19); (2) lupeol (20); and (3) betulinic acid (21) were acquired based on comparing their spectroscopic data (NMR and EI-MS) with those described in the literature (Table 1). The NMR elucidation procedures have been described in the supplementary file.
Table 1.
NMR Spectroscopic Data for (1) Taraxasterol; (2) Lupeol; and (3) Betulinic Acida
| Taraxasterol | Lupeol | Betulinic Acid | ||||
|---|---|---|---|---|---|---|
| Number | δH (J in Hz) | δC | δH (J in Hz) | δC | δH (J in Hz) | δC |
| 1 | - | 39.1 | - | 38.9 | - | 39.1 |
| 2 | - | 27.5 | - | 25.6 | - | 27.8 |
| 3 | 3.21, dd (6.4, 13.1) | 79.3 | 3.24, dd (4.8, 11) | 79.0 | 2.98, dd (5.9, 10) | 78.3 |
| 4 | - | 38.9 | - | 39.6 | - | 39.1 |
| 5 | - | 55.4 | - | 55.4 | - | 55.6 |
| 6 | - | 18.5 | - | 18.3 | - | 18.6 |
| 7 | - | 34.3 | - | 34.0 | - | 34.7 |
| 8 | - | 50.0 | - | 41.2 | - | 40.3 |
| 9 | - | 50.5 | - | 50.5 | - | 50.0 |
| 10 | - | 37.2 | - | 37.1 | - | 37.5 |
| 11 | - | 21.7 | - | 21.5 | - | 21.0 |
| 12 | - | 26.4 | - | 27.4 | - | 25.8 |
| 13 | - | 39.1 | - | 39.4 | - | 38.3 |
| 14 | - | 42.3 | - | 42.9 | - | 42.5 |
| 15 | - | 26.7 | - | 27.6 | - | 30.7 |
| 16 | - | 38.6 | - | 38.3 | - | 32.6 |
| 17 | - | 34.5 | - | 44.1 | - | 56.5 |
| 18 | - | 47.1 | - | 48.7 | - | 47.0 |
| 19 | 2.37 m | 40.1 | 2.25 m | 47.6 | 2.82 m | 49.5 |
| 20 | - | 154.9 | - | 151.1 | - | 150.3 |
| 21 | - | 25.9 | - | 29.7 | - | 30.0 |
| 22 | - | 39.0 | - | 40.0 | - | 37.5 |
| 23 | 0.78 s | 28.4 | 1.00 s | 28.0 | 0.68 | 28.0 |
| 24 | 0.84 s | 15.6 | 0.78 s | 15.4 | 0.74 | 15.4 |
| 25 | 0.86 s | 17.1 | 0.88 s | 16.4 | 0.72 | 16.3 |
| 26 | 0.90 s | 16.2 | 1.02 s | 15.9 | 0.75 | 16.5 |
| 27 | 0.94 s | 14.9 | 0.93 s | 14.8 | 0.78 | 14.7 |
| 28 | 0.85 s | 18.7 | 0.86 s | 17.8 | - | 179.6 |
| 29 | 1.02 s | 26.0 | 1.62 s | 19.5 | 1.50 | 19.7 |
| 30 | 4.62 s | 107.4 | 4.63 s | 107.2 | 4.40 s | 109.2 |
| 4.64 s | 4.65 s | 4.53 s | ||||
a1H-13C NMR data recorded in CDCl3; (400 MHz for δH, 100 MHz for δC)
The taraxastane and lupane triterpenes have exhibited the potential cytotoxicity (26-29). Hence, to determine the cytotoxic activities of isolated triterpenoids (1-3), cellular toxicity was evaluated by MTT assay on two cancerous cell lines - the human gastric carcinoma (AGS) cell line as well as the mouse skin fibrosarcoma (WEHI-164) cell line. The toxicity effect at 0 - 200 µM concentrations was detected in a time- and dose-dependent manner. As shown in Table 2, the results of cytotoxicity revealed that compounds 2 and 3 with lupane structures possess stronger activity than compound 1 with taraxastane framework. Interestingly, compounds 1 - 3 in particular were more active than the positive control compound, dendrosmal curcumin, with an expectation for compound 1 on WEHI-164 cell line behaving a less actively than that of dendrosmal curcumin (IC50 values of 23.8 and 18.0 µM, respectively). Although some other similar structures have been previously reported from other Chrysanthemum species, for instance, arnidiol, faradiol (taraxastane-type), calenduladiol and heliantriol B2 (lupane-type) from C. morifolium. Arnidiol had remarkably exhibited a wide range of cytotoxicity against sixty human cancer cell lines (GI50 values of mostly < 6 μM) (13).
Table 2.
Cytotoxic Activity of Compounds 1 - 3 Isolated from the T. disciforme Aerial Parts
| Natural Compounds | IC50, µMa | |
|---|---|---|
| AGS | WEHI-164 | |
| Taraxasterol (1) | 16.1 ± 0.4 | 23.8 ± 2.1 |
| Lupeol (2) | 10.4 ± 1.7 | 3.2 ± 0.9 |
| Betulinic acid (3) | 5.6 ± 1.3 | 7.7 ± 1.6 |
| Dendrosmal curcuminb | 16.6 ± 0.6 | 18.0 ± 0.8 |
aThese data represent the average values of three repeated experiments and expressed as mean ± SD.
bDendrosmal curcumin was used as a positive control; the values are checked in every experiment.
Molecular docking studies of three metabolites-taraxasterol, lupeol and betulinic acid were carried out with Bcl-2, Bcl-xl, CDK2 accompanied by tubulin proteins using AutoDock Vina, to recognize the binding mode of ligands and the intramolecular hydrogen bond and other interactions between the target proteins and ligands. The outcomes revealed that the best interactions were related to lupeol and/or taraxasterol with Bcl-xL. On the other hand, the weakest interaction was related to betulinic acid with Bcl-2. The interactions of isolated compounds with tubulin recorded by computational docking study have been presented in Figure 2A. The schematic 2D plots were displayed intermolecular interactions of the representative taraxasterol with tubulin (Figure 2B). The Bcl-2 protein functions to prevent apoptosis through protection of mitochondrial external membrane integrity via binding to Bax/Bak (30). The high binding affinity of compounds 1 - 3 indicated the ability to either inhibit the Bcl-2 or induce the apoptosis trend (mentioned in the Appendix 1 in Supplementary File).
Figure 2.
A, Binding modes at: vinblastine binding site of tubulin (PDB: 4eb6), betulinic acid (green), lupeol (pink), taraxasterol (blue); B, hydrophobic interactions and hydrogen bonding between taraxasterol and tubulin.
Note that overexpression of the antiapototic protein, Bcl-xL, provides a public mechanism bringing about the survival of cancerous cells along with special resistance to the conventional chemotherapy. Therefore, an attractive strategy for cancer therapy was proposed as the specific enzyme inhibition (31). In a respective manner, taraxasterol, lupeol and betulinic acid have had potent interaction with Bcl-xL along with the high binding affinity -8.9, -9.3 and -9.3 kcal/mol (Figure 3).
Figure 3.
A, Proposed binding model of betulinic acid (green), lupeol (pink), taraxasterol (blue) within the Bcl-xL (PDB: 3spf); B, hydrophobic interactions and hydrogen bonding between lupeol and Bcl-xL.
Additionally, the cycling-dependent kinase-2 (CDK2) plays a key role in regulating several events of eukaryotic cell division cycle. The evidence demonstrated that over expression of CDK2 might be a main reason of the abnormal regulation of cell-cycle, which would be straightly related to hyperproliferation in cancer cells. The microtubules formed during polymerization of α- and β-tubulins; however, the most important role of microtubules is to create the mitotic spindle involved in cell division (32). It has been reported that the afforded triterpenoids not only could effectively decrease the levels of Bcl-2 expression but upregulate the box gene in various cells as previously approved (27, 31, 33). Other studies have discussed how these compounds could be act as the main factor of downregulation in Bcl-xl and CDK2 expression (34, 35). In the present study, our data indicated that the compounds 1 - 3 could computationally inhibit Bcl-2 protein activity and impact on the prevention of tubulin polymerization because of the strong interaction existing between all compounds with the vinblastine binding site of tubulin.
4.1. Conclusions
To the best of our knowledge, this is the first study of isolation and structure elucidation of three known triterpenes (taraxasterol, lupeol and betulinic acid) from dichloromethane extract of the Tripleurospermum disciforme (C. A. Mey.) Schultz-Bip. aerial parts, elucidated by spectroscopic analyses in comparison with spectroscopic and physical data mentioned in the literature. As these isolated compounds exert the ability of cytotoxic activity studied by computational technique (molecular docking), its future pharmacological application is of particular importance because it may be consequential in the prevention of cancer.
