Hydrodistillation of the freshly chopped rhizome of Curcuma xanthorrhizza Roxb. yielded 0.44% of essential oil. GC-MS results showed that xanthorrhizol is the major chemical constituent in the essential oil as it makes up nearly 46.3% of the total component (Figure 1A). Compound 1 was obtained as light brownish oil and was given name as xanthorrhizol. TLC profile of 1 presented the R_f value of 0.27 using 5% diethyl ether in petroleum ether (Figure 1B, 1C). Meanwhile, TIC profile showed an integrated single peak of 1 with 100% peak area (CAS# 30199-26-9, Figure 1D). Its molecular formula was obtained from high resolution mass spectrum which showed [M⁺] ion at m/z 218.3210 corresponding to the formula C₁₅H₂₂O; calc 218.3404 (Figure 2). In ¹H NMR analysis, the presence of phenyl group was evident from signals at δ(ppm) 6.63 (s, 1H), 6.71 (dd, 1H, J = 7.5, 1.8, 1.5 Hz) and 7.05 (d, 1H, J = 7.8 Hz) in the ¹H NMR spectrum (Figure 1E).

Compound 1 (xanthorrhizol): Light brownish oil; C15H22O; High MS m/z: M+ 218.3210 calc. 218.3404 for C₁₅H₂₂O; Low MS m/z (%): 218(42), 48 (28), 136 (100), 121 (57), 135 (54); 1H NMR (300 MHz, CDCl₃): δ 1.22 (d, 3H, J = 6.9 Hz, -CH₃-6), 1.54–1.63 (m, 2H, -CH₂-4), 1.70 (s, 6H, -C(CH₃)₂-1), 1.87–1.94 (m, 2H, -CH₂-3), 2.24 (s. 3H, -CH₃-10), 2.57–2.69 (m, 1, -CH-5), 5.08–5.14 (m, 1H, -CH = C-2), 6.63 (s, 1H, Ph), 6.71 (dd, 1H, J = 7.5, 1.8, 1.5 Hz, Ph), 7.05 (d, 1H, J = 7.8 Hz, Ph)

Simultaneous treatment of fixed combination ratio with an increasing fraction of curcumin has showed a synergistic cytotoxic effect in MDA-MB-231 cells as revealed by the isobologram and combination indexes method (Figure 3A, 3B). Synergistic activity was commenced from the combination xanthorrhizol-curcumin 3:7 to 1:9. These data reflects the vital involvement of curcumin in the sensitiviy of MDA-MB-231 cells towards the test agents.

We had conducted two types of sequential treatments where xanthorrhizol or curcumin was used to treat the MDA-MB-231 cells for 24 hours, followed by the removal of total medium and replenished again with curcumin- or xanthorrhizol-added medium at a fixed combination ratio as in the simultaneous treatment for further 24 hours. The first treatment (X-C) showed antagonism at lower fraction of xanthorrhizol but approaching additivity when the xanthorrhizol was at higher fraction (Figure 3C, D). In contrary, the latter treatment (C-X) resulted in synergistic effect at higher fraction of curcumin and this trend was quite similar with cells that are treated simultaneously with xanthorrhizol and curcumin (Figure 3E, F). Results from both sequential treatments imply the cytotoxicity of the combined treatment of xanthorrhizol and curcumin was schedule-dependent and curcumin may plays a major role in enhancing the cytotoxicity of xanthorrhizol in the MDA-MB-231 cells. Hence, simultaneous treatment was suggested as the better treatment route as compared to both the sequential treatments.

The cationic dye MitoCapture™ was able to enter the cell cytoplasm, accumulate and aggregate in mitochondria, thus producing a bright red fluorescence. This red fluorescence was easily distinguished in untreated MDA-MB-231 cells. Remarkably, increasing doses of xanthorrhizol-curcumin simultaneous treatment resulted in significant reduction of this red fluorescence. On the other hand, there were an increasing number of treated cells which only fluoresces green. This is due to the inability of MitoCapture™ to accumulate in mitochondria as the mitochondrial transmembrane potential alters during apoptosis events. They remain as green monomers in the cytoplasm of apoptotic cells. A similar trend of fluorescence was also seen in MDA-MB-231 cells when treated with 15 μg/ml of xanthorrhizol (X) and 5 μg/ml and 10 μg/ml tamoxifen (TAM) respectively (Figure 2A).

The Hoechst 33258 dye was able to diffuse through intact membranes of MDA-MB-231 cells and stain their DNA. As the concentration of combined xanthorrhizol-curcumin increased, single intense fluorescence and multiple strong fluorescence signals were produced in the cell nuclei (Figure. 2B). The control culture (untreated MDA-MB-231) was uniformly stained with no substantial fluorescence signal, whereas the treated cells showed clear apoptotic morphology. According to the fluorescence and phase contrast images, shrinkage of cells and plasma membrane convolution were all observed in the treated cells. Similar apoptotic morphology was also observed in the MDA-MB-231 cells treated with 15 μg/ml xanthorrhizol (X). The number of apoptotic cells was determined and expressed as percentage of apoptotic index. Simultaneous treatment with combined xanthorrhizol-curcumin resulted in a dose dependent increase in apoptotic cells (Figure 4).

DNA fragmentation is a biochemical hallmark of apoptotic cell death. From the agarose gel, DNA samples from the treated MDA-MB-231 cells exhibit intact genomic DNA (gDNA) and clear DNA ladders during treatment at higher doses (Figure 5). Treatments at lower doses showed only intact gDNA and do not marked any DNA laddering or even smearing effect. When the multiple bands were analyzed, they showed ~180 bp inter-nucleosomal excision, thus confirming apoptosis but not necrosis had taken place. Lane T and C consist of DNA sample from taxol-treated MDA-MB-231 cells and actinomycin-treated HL 60 cells respectively as positive control. The same pattern of DNA laddering was observed.

Fresh rhizomes of C. xanthorrhizza Roxb. were chopped and essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus. Distilates were then extracted 3 times with diethyl ether. The pooled organic phases were dried and subjected to two rounds of silica gel vacuum column chromatography (VCC) separation (Merck silica gel 60, 0.040–0.063 mm; and Merck silica gel 60, 0.063–0.200 mm respectively). The columns were eluted with petroleum-ether with gradual increase ratio of diethyl ether. The existence of xanthorrhizol in each fraction was pre-identified using thin layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) by comparing the retention factor (R_f) in TLC profile and the total ion chromatogram (TIC)/Mass spectra in GC-MS profile respectively with those of authentic reference substance (Alexis Biochemicals, Switzerland). Fractions were subjected to GC-MS analysis on an Agilent system with a model 6890 gas chromatography, a model 5073 Mass Selective detector (MSD) and an Agilent Chemstation data system. The following GC oven temperature program was used: 40°C initial temperature; held for 2 min; increased at 5°c/min to 230°C with split (50:1) injection.

From our previous study, xanthorrhizol is not able to be separated by normal CC using mixture of solvents with different polarity as other compounds always appeared together as co-eluents. In an effort to purify xanthorrhizol from the final fraction from VCC, subsequent reaction steps (Scheme 1 and Scheme 2) were conducted to facilitate the isolation and purification. Xanthorrhizol was reported as a potential chiral starting material for the synthesis of other sesquiterpenoids 43. As a result, we started the process of acetylation towards fractions that comprise compound 1 and other co-eluents to produce a lower polarity compound 2 namely, acetyl-xanthorrhizol (Scheme 1).

This particular fraction which consists of compound 2 and other components was subjected to open CC to separate compound 2 and other compounds. Compound 2 was easily separated from other compounds in this step as its polarity was increased after the acetylation process. Following fractions which contain only 2 were then pooled and undergone hydrolysis to produce back xanthorrhizol (1), as showed in scheme 2. Subsequent partition was then able to separate 1 into organic phases in EtOAc. Finally, evaporation of the organic solvent yielded 1 as a light brownish oil with 71% yield.

Identification of 1 was carried out by comparing its GC mass spectral to the authentic reference substance (Alexis Biochemicals, Switzerland), referring to the NIST98 database/Chemstation data system (National Institute of Standards and Technology, Gaithersburg, MD, USA). Subsequent identification was conducted by using ¹H-NMR analysis. NMR spectral (300 MHz) of 1 was recorded on a Bruker Avance 300 Spectrometer using CDCl₃ as solvent. Chemical shifts are reported in ppm with reference to residual solvent [CHCl₃ (7.26)].

MDA-MB-231 human breast cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). They are maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen Co., Carlsbad, CA, USA) supplemented with 5% heat-inactivated fetal calf serum (Invitrogen Co.), 100 U/ml penicillin and 100 mg/ml streptomycin (Flowlab, Sdyney, Australia) in a humidified atmosphere of 5% CO₂ in air at 37°C. Cells were kept in the logarithmic growth phase by routine passage every 2–3 days using 0.025% trypsin-EDTA treatment.

The antiproliferative activity of fixed ratio combination of xanthorrhizol and curcumin (from 1:9 to 9:1) towards MDA-MB-231 human breast cancer cell line was evaluated by using the sulforhodamine B (SRB) method, as previously described 5859. Cells were seeded 24 hours prior to treatment in a 96-well plate at plating densities 15, 000 cells/well in order to obtain semi-confluent cultures. One plate from the cell line was fixed in situ with TCA (Sigma Chemical Co.), to represent a measurement of the cell population for each cell line at the time of drug addition (T_z). Combination solutions were dissolved in DMSO (Sigma Chemical Co.) and followed by a 2× serial dilution for 6 points ranged from 40 μg/ml to 1.25 μg/ml. The final concentration of DMSO used in the corresponding wells did not exceed 1% (v/v), which affect cell viability. Following drug addition, the plates were incubated for an additional 48 hours.

At the end of incubation, cells were fixed in situ with 50 μl of cold 50% (w/v) TCA and incubated for 1 hour at 4°C. The plates were then washed with tap water and air dried. SRB (Sigma Chemical Co.) solution (100 μl) at 0.4% (w/v) in 1% acetic acid was added to the corresponding wells and further incubated for 10 minutes at room temperature. After staining, unbound dye was washed out by 1% acetic acid and the bound stain was subsequently solubilised with 10 mM trizma base (Sigma Chemical Co.). The absorbance at 505 nm was read on a spectrophotometric plate reader.

Dose-response curves were constructed to obtain the response parameter which was the GI₅₀. Percentage growth is calculated as: [(T_i - T_z)/(C - T_z)] × 100 where (T_z) is time zero, (C) is control growth and (T_i) is test growth in the presence of drug at the six concentration levels. The GI₅₀ value (growth inhibitory activity) corresponds to the concentration of test agents causing 50% decrease in net cell growth which was calculated from [(T_i - T_z)/(C - T_z)] × 100 = 50. All data were derived from 3 independent experiments.

Drug synergy was determined by the isobologram analysis and combination-index (CI) methods, as described elsewhere 6061. Isobologram analysis was used to determine the effects of combinations of drugs on MDA-MB-231 cells whereas the CI method is a mathematical and quantitative representation of two-drug pharmacologic interaction. Using data from the growth inhibition assay, classic isobologram was constructed and CI values were then calculated by using the following formula: CI = (d1/D_x1) + (d2/D_x2), where D_x is the dose of one drug alone required to produce a 50% growth inhibition, and d1 and d2 are the doses of xanthorrhizol and curcumin respectively, in combination that produce the same effect (50% growth inhibition). A CI of > 1 implies antagonism, = 1 is additivity, and < 1 is synergy.

Changes in the mitochondrial membrane potential of treated MDA-MB-231 cells were examined using a fluorescent microscope (Imaging Source Europe GmbH) and MitoCapture™ Mitochondrial Apoptosis Detection Kit (BioVision Research, Moutain View, CA, USA), according to the manufacturer's instructions. Briefly, treated and untreated cells were washed with PBS. One μl of MitoCapture dye was diluted to 1 ml pre-warmed incubation buffer immediately prior to use. The cells were then incubated in 500 μl of the diluted MitoCapture solution at 37°C for 20 minutes in a CO₂ incubator before being observed under a fluorescence microscope using a band-pass filter.

Nuclear staining with Hoechst 33258 (Sigma Chemical Co.) was performed as described elsewhere 362. Briefly, treated and untreated cells were washed with phosphate buffered saline (PBS) followed by incubation in 10 μM Hoechst 33258 solution at room temperature for 20 min. At the end of incubation, cells were washed extensively with PBS. Nuclear morphology was then examined under a fluorescence microscope (Imaging Source Europe GmbH, Bremen, Germany). To quantify the apoptotic index, the percentage of single and multi intense-fluoresced cells (apoptotic morphology) were calculated from five random microscopic fields at ×40 magnification.

DNA fragmentation assay is a definitive assay to confirm apoptotic cell death has taken place in the cultures. DNA fragmentation assay was conducted using the Suicide Track™ DNA Ladder Isolation Kit (Calbiochem), according to manufacturer's instructions. Briefly, MDA-MB-231 cells were cultured in a T-25 flask and treated with combined solution of xanthorrhizol-curcumin for 24 hours. Floating and and trypsinized-adherent of treated and untreated cells were collected by centrifuging at 900 × g for 5 min. Cell pellets were then resuspended in 250 μl of extraction buffer following incubation on 4°C for 30 min. The suspensions were then centrifuged at 16, 000 × g for 5 min at room temperature. The resulting supernatant were transferred to a clean tube, followed by the addition of 10 μl of RNase A solution and incubated at 37°C for 1 h. DNA isolation buffer was added and further incubated at 50°C for overnight. During the DNA precipitation step, 2 μl of Pellet Paint^® Co-precipitant was added to the suspension together with 30 μl of 3 M sodium acetate and 311 μl of 2-propanol. Samples were mixed by inversion and then centrifuged at 16, 000 × g for 5 min. The resulting pellets were washed with 70% and 100% ethanol. The DNA samples were then air-dried and resuspended in 50 μl of resuspension buffer (10 mM Tris, 1 mM EDTA). Finally, DNA samples were separated in 2% agarose gel in 1× TAE. Positive control was supplied by Calbiochem which consist of 1 × 10⁶ HL60 cells treated with 0.5 mg/ml Actinomycin D for 19 h.

All data were expressed as mean ± standard deviation. The statistical differences were analyzed using one-way ANOVA followed by a Tukey Honestly Significantly Different (HSD) test. Values of p < 0.05 were considered significant.