Bioactive compounds from Curcuma aeruginosa and the effect of comosone II on the migration and invasion of breast cancer cells

Abstract Bioassay-guided separation afforded furanodienone 1,10-epoxide (9) as the new compound, curcolone (10) as partially described compound and ten known compounds; germacrone (1), furanodienone (2), curzerenone (3), curcumenol (4), zederone (5), comosone II (6), (1E,4E,8R)-8-hydroxygermacra-1(10),4,7(11)-trieno-12,8-lactone (7), 13-hydroxygermacrone (8), curcuzederone (11) and demethoxycurcumin (12). The study showed that germacrone, furanodienone, curzerenone, comosone II, 13-hydroxygermacrone, curcuzederone and demethoxycurcumin are the bioactive compounds of C. aeruginosa rhizomes. Comosone II significantly inhibited MDA-MB-231 cell migration and invasion through the inhibition of MMP-9 enzyme. The present study may lead to further anticancer studies of comosone II and supports the traditional uses of C. aeruginosa rhizomes. Graphical Abstract


Introduction
Curcuma aeruginosa (Family: Zingiberaceae) is commonly found in Malaysia, Indonesia, India, and Bangladesh. Rhizomes are popular as the traditional medicines for the treatment of asthma, tumor, cancer, uterine pain and uterine inflammation [1][2][3][4]. The pharmacological studies reported from the species are antioxidant, antinociceptive, anti-inflammatory and antimicrobial activities [2,5]. The antimigratory activity of different extracts of C. aeruginosa rhizomes against the MDA-MB-231 cell line was also reported [6].
Based on the traditional uses and the reported studies, the present study was carried out to isolate bioactive compounds from C. aeruginosa rhizomes and investigate the effect of comosone II (6) on the migration and invasion of highly invasive TNBC cell line MDA-MB-231.

Results and discussion
The inhibitory activity on the viability of MDA-MB-231 cells was evaluated using the cell viability assay. The crude extract showed an IC 50 value of 135.1 mg/ml and the most potent chloroform fraction showed an IC 50 value of 64.9 mg/ml ( Table 1).
Furanodienone 1,10-epoxide (9) was obtained as the colorless solid. Both the 13 C-NMR data and HRESIMS measurement supported the molecular formula C

15
H 18 O 3 . The presence of a conjugated carbonyl group was evident from a C ¼ O stretch at  (2) 274.6 mM Curzerenone (3) 298.0 mM Curcumenol (4) N A Zederone (5) NA Comosone II (6) 243.5 mM (1E,4E,8R)-8-hydroxygermacra-1(10),4,7(11)-trieno-12,8-lactone (7) N A 13-hydroxygermacrone (8) 232.3 mM Furanodienone 1,10-epoxide (9) NA Curcolone (10) N A Curcuzederone (11) 227.2 mM Demethoxycurcumin (12) 124 showed HMBC cross-peaks with d C 58.9 (q), 69.9 (t) and 40.2 (s). The appearance of a high field shifted oxygenated quaternary carbon atom (d C 58.9) and the presence of another oxygenated tertiary carbon atom (d C 69.9) in nearby position suggested the presence of an epoxy group in 9. The spectral data of 9 (Table 2) are very similar with those of isolated furanodienone (2). Compound 9 has an additional epoxy ring at C 1 -C 10 carbon atoms instead of a double bond in those carbon atoms of furanodienone (2). We, therefore, proposed the name of 9 as furanodienone 1,10-epoxide. The relative configuration of C-1 and C-10 chiral carbon atoms was studied by the NOESY experiment. H-1 at d H 2.96 (dd), did not show a cross peak with CH 3 -15 at d H 1.02, s, in the NOESY experiment ( Figure 3). The peaks of H 3 -15 at d H 1.02 and H a -9 at d H 3.59 showed cross-peaks with H a -9 at d H 3.59 and H b -9 at d H 2.72, respectively. These cross-peaks clearly revealed the a orientation of the epoxy group at C-1 carbon and b orientation of the epoxy group at C-10 carbon ( Figure 3). These findings are also consistent with the biogenic perspective as it is derived from furanodienone. Moreover, the compounds showed [a] D À 2.93 (c ¼ 1.0) which supported a orientation at C-1 carbon atom and b orientation at  Table 2. The 1 H-NMR (500 MHz) and 13 C-NMR (125 MHz) spectral data of compounds 9 and 10 in CDCl 3 . 9 1 0 9 1 0 C-10 carbon atom of the epoxy group. As compound 9 is identical to furanodienone (2) except for the epoxy group at C-1 to C-10, therefore, it was named furanodienone 1,10-epoxide (9). Curcolone (10) was obtained as the colorless solid. The 1 H-NMR, 13 C NMR (Table 2), DEPT 135, DEPT 90, COSY, HSQC and HMBC spectroscopic data were further used to elucidate the chemical structure. The chemical structure was identical to curcolone reported in the literature [13]. However, the previous study elucidated the structure from UV, IR and 1 H-NMR data. The study confirmed the absolute configuration from the splitting patterns of proton signals in 1 H-NMR spectrum and the biogenetic point of view by comparison with zederone. In this study, the structure of curcolone (10) was elucidated from 1 D and 2 D NMR spectral data and the NOESY spectrum confirmed the similarity of curcolone configuration with the one reported in the literature [13].
Comosone II (6) is a sesquiterpenoid isolated from some species of Zingiberaceae. The inhibitory activity of comosone II (6) on the viability of MCF-7, Ca Ski, PC-3, HT-29 and HUVEC cell lines was reported [8]. However, there is no study against TNBC cells MDA-MB-231 in the literature. Therefore, the present study was carried out to investigate the effect of comosone II (6) on the migration and invasion of MDA-MB-231 cells by using the scratch, transwell migration and invasion assays. The results of the scratch assay showed 22.2%, 29.2% and 45.8% inhibitions at the concentrations of 50, 100 and 150 mM, respectively ( Figure 4). The transwell migration assay showed significant concentration-dependent inhibition of MDA-MB-231 cell migration by comosone II (6) at the concentrations tested ( Figure 5). Comosone II (6) in transwell invasion assay showed a concentration-dependent and significant reduction of MDA-MB-231 cell invasion at three selected concentrations ( Figure 6).
The effect of comosone II (6) on MMP-2 and MMP-9 activities was evaluated in gelatin zymography. In this experiment, the inhibitions of the MMP-9 enzyme by comosone II (6) at the concentrations of 100 and 150 mM were 32.7% and 48.8%, respectively (Figure 7). Secretions of MMP-2 and MMP-9 in the media by MDA-MB-231 cells treated with comosone II (6) were measured by ELISA. As shown in Figure 8, comosone II (6) significantly reduced the secretion of the MMP-9 enzyme to 74.0% and 58%, at the concentrations of 100 mM and 150 mM, respectively. The findings of the present study may lead to further molecular mechanism and in-vivo studies of comosone II for potential drug development and provide evidence-based support for the traditional uses of C. aeruginosa rhizomes for cancer.

Plant material
Rhizomes (20 kg) were collected from Panchagarh, Bangladesh. The plant was identified by Ms. Hosne Ara, the Director of Bangladesh National Herbarium,and a specimen was deposited with the Accession Number of 37514.

Extraction and isolation
The dried rhizomes were ground to get 2.8 kg powder. The powder (2.0 kg) was macerated in methanol (2.0 kg Â 5 L Â 72 h, 3 times) and drying of the extracting    (6). Results are the mean ± SEM of three independent experiments. Ã p < 0.05 and ÃÃ p < 0.01 when compared to the control. solvent was carried out in a rotary evaporator which yielded 220 g of the crude extract.

Cell viability assay
MDA-MB-231 cells were obtained from ATCC, Rockville, MD, USA. The activity of the extract, fractions, and compounds on the viability of MDA-MB-231 cells was assayed following the previously described method [17] 3.5. Scratch assay The effect of comosone II (6) on the migration of the MDA-MB-231 cell line was evaluated by the scratch assay as described previously [17].

Transwell migration assay
The migration inhibitory activity was further investigated by the transwell migration assay. The MDA-MB-231 cells (30 Â 10 4 cells/ml) were suspended in DMEM and treated with comosone II and 0.1% DMSO for 30 min and the experiment was further carried out following the method described previously [17] 3.7. Transwell matrigel invasion assay The effect of comosone II on the invasion of MDA-MB-231 cells was investigated by the transwell matrigel invasion assay. The upper inserts of polycarbonate filters (8 lm) were coated with 100 ml matrigel solution (1:8 dilution, 920 mg/ml) and incubated for 2.5 h. The MDA-MB-231 cells (6.0 Â 10 5 cells/ml) were suspended in the medium (2.5% FBS in DMEM) treated with comosone II and the experiment was further carried out following the procedure as described in the transwell migration assay [17].

Gelatin zymography
The effect of comosone II on MMP-2 and MMP-9 activities was investigated by using the gelatin zymography. Briefly, MDA-MB-231 cells (10 Â 10 4 cells/well) were seeded into a 6-wells plate and incubated for 24 h. The cells were then treated with comosone II in the serum-free DMEM and incubated for 24 h. The detection of MMP-2 and MMP-9 enzymes was carried out following the previously described method [17].
3.9. Measurement of MMP-2 and MMP-9 concentrations by ELISA MDA-MB-231 cells (10 Â 10 4 cells/well) were seeded in a 6-well plate and incubated for 24 h. The cells were treated with serum free DMEM containing comosone II and incubated for 24 h. Following incubation, the media were collected, centrifuged and the supernatant was concentrated. The MMP-2 and MMP-9 concentrations were measured using MMP-2 and MMP-9 immunoassay kits according to the manufacturer's protocol.

Statistical analysis
The data were presented as the mean ± SEM of three independent experiments. Oneway ANOVA followed by Dunnett's multiple comparison tests was used to determine the significance.

Disclosure statement
The authors declare no conflicts of interest in this study.