Costunolide and parthenolide from Champi Sirindhorn ( Magnolia sirindhorniae ) inhibit leukemic cell proliferation in K562 and Molt-4 cell lines

The magnolia plant has been used in traditional medicine since ancient times. This study was designed to investigate the effects of active compounds from Thai Champi Sirindhorn ( Magnolia sirindhorniae ) on leukemic biomarker Wilms ’ tumor 1 ( WT1 ) protein expressions in K562 and Molt-4 cells . Costunolide ( 1 ) and parthenolide ( 2 ) were the major components used in this study, they were purified from ethyl acetate fractions . Costunolide ( 1 ) and parthenolide ( 2 ) had strong cytotoxic effects in K562 and Molt-4 cells measured with MTT assays . Their activities were compared to standard commercial costunolide ( 3 ) and parthenolide ( 4 ) . Costunolide ( 1 ) and parthenolide ( 2 ) decreased WT1 protein levels and total cell numbers in K562 and Molt-4 cells . Both purified costunolide ( 1 ) and standard commercial costunolide ( 3 ) decreased WT1 protein levels in a time - and dose - dependent manner . Therefore, the active compounds from M . sirindhorniae were identified as promising sources for bioactive compounds for further applications in traditional medicine.

Trypan blue dye solution was purchased from AMRESCO (Solon, OH, USA). Rabbit polyclonal anti-WT1 antibody  and rabbit polyclonal anti-GAPDH antibody were purchased from Santa Cruz Biotechnology (CA, USA), and HRP conjugated goat antirabbit IgG was purchased from Invitrogen Life (Carlsbad, CA, USA). Enhanced chemiluminescence detection kit was purchased from Thermo Scientific (Miami, USA), and Luminata Forte Western HRP Substrate was purchased from Millipore Corporation (Billerica, MA, USA). n-Hexane, ethyl acetate, and methanol were purchased from Merck (Darmstadt, Germany).

Plant materials
The leaves, twigs, and stems from the M. sirindhorniae plant (over than 3 years old) were collected from the Thailand Institute of Scientific and Technological Research, Khlong Luang District, Pathum Thani Province, Thailand, in April 2010. A voucher specimen No. BKF420621 was deposited at the herbarium in the Royal Forest Department, Ministry of Agriculture and Cooperatives, Bangkok, Thailand. The herbarium specimen has been studied and annotated with traditional methods of herbarium taxonomy.

Extraction and isolation of M. sirindhorniae
Nine crude fractional extracts were prepared utilizing different solvents for the various parts of the plant. Specifically, fractions No. 1 -3 were extracted from the leaves of M. sirindhorniae, fractions No. 4 -6 from twigs, and fractions No. 7 -9 from the stems. The fractions were extracted by using n-hexane (Hex), ethyl acetate (EtOAc), and methanol (MeOH), respectively. In addition, the active fractions were purified with column and thin layer chromatography (TLC) until the purified compounds 1 and 2 were obtained. The two compounds (1 and 2) were identified as costunolide (1) and parthenolide (2) (Tiuman et al. 2005;Katekunlaphan et al. 2014;Venditti et al. 2016). To identify the compounds, spectroscopic analyses, including infrared spectroscopy, electrospray ionization-mass 4 spectrometry, and 1 H and 13 C nuclear magnetic resonance (NMR), were performed. Twodimensional-NMR measurements, such as correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC), also supported the identifications of these compounds (Figures s1 -s18). The structures for costunolide (1) and parthenolide (2) are shown in Figures 1(a) and 1(b), respectively.

Cells and cell culture conditions
The K562 and Molt-4 cell lines, a model of WT1-overexpressing leukemic cells, were cultured in a RPMI-1640 medium supplemented with a 10% fetal bovine serum, 1 mM Lglutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin. They were then incubated under the following conditions, a 95% relative humidity with 5% CO 2 at 37 C.

MTT cytotoxicity assay
The cytotoxicity of the crude fractional extracts (fractions No. 1 -9), costunolide (1), and parthenolide (2) were evaluated using MTT assays. Briefly, the K562 and Molt-4 cells (1.0 × 10 4 cells/well) were cultured in 96 well plates containing 100 µL medium prior to treatment for 24 h. After that, 100 µL of fresh medium containing various concentrations (0-100 µg/mL) of the test compounds were added to each well and incubated for 48 h. The MTT dye solution was added (15 µL/100 µL medium) and the plates were incubated at 37 C for 4 h in a humidified 5% CO 2 conditions. Afterwards, 200 µL of DMSO was added to each well, and mixed thoroughly to dissolve the dye crystals. The absorbance was measured using an ELISA plate reader (Biotek EL 311) at 570 nm with a reference wavelength of 630 nm. High optical density readings corresponded to a high intensity of dye color, representing a high number of viable cells able to metabolize MTT salts. The fractional absorbance was calculated with the following formula: The average cell survival obtained from triplicate determinations at each concentration was plotted as a dose response curve. This was completed in three independent experiments.
The 50% inhibitory concentrations (IC 50 ) of the active substances were determined to be the 5 lowest concentrations which reduced cell growth by 50% in those treated compared to untreated cultures or vehicle control cultures (0.2% DMSO in culture medium). The IC 50 values are representative of the activities and are shown as a mean ± standard deviation (SD).

Trypan blue exclusion test
Cell proliferation was measured by using the Trypan blue exclusion method. Cells were treated with various concentrations of crude fractional extracts (fractions No. 1 -9), purified active compounds (costunolide (1) and parthenolide (2)), and commercial compounds (costunolide (3) and parthenolide (4)). Then, cells and 0.4% Trypan blue dye were mixed together and counted using a light microscope. All experiments were performed in triplicate.

Protein extraction and Western blotting
K562 and Molt-4 cells were treated with crude fractional extracts (fractions No. 1 -9), the purified active compounds (costunolide (1) and parthenolide (2)), and commercial compounds (costunolide (3) and parthenolide (4)) for 48 h, after which the cells were collected, washed twice with a cold PBS, and lysed with a cold RIPA buffer (50 mM Tris, 0.1% SDS, 1% Triton X-100, 150 mM NaCl, 0.5 mM EDTA, and 0.001% protease inhibitor cocktail) for whole protein extraction. Whole protein lysates (30 µg) were loaded onto 12% SDS-PAGE and then transferred to PVDF membranes (Merck and Millipore, Burlington, MA, USA). Membranes were blocked with 5% skim milk and probed with rabbit anti-WT1 at a dilution ratio of 1:1,000. Rabbit anti-GAPDH at a dilution ratio of 1:1,000 was used for the protein loading control. The reaction was followed by HRP-conjugated anti-rabbit IgG at 1:20,000 dilution.
Proteins were shown using an enhanced chemiluminescence detection kit, and Luminata Forte Western HRP Substrate. The chemiluminescent signals were detected using a Typhoon TRIO Imager. Densitometry was performed using the Alpha Innotech software. The band density of the loading control was used to normalize the band densities of the proteins of interest to obtain the relative normalized expression level as compared to the exposed control.

Chromatographic analysis
The fractions for No. 1, 2, 3, and standard costunolide (3) were weighed and dissolved in methanol at the final concentration of 0.0808 mg/mL. Then the mixture was filtered through a 0.45 µm syringe filter and analyzed by HPLC (Agilent 1100 DAD). The HPLC system consisted of a C-18 reversed-phase column (Verticep usp 150L 4.6 mm). The costunolide was eluted with a mobile phase consisting of methanol and distilled deionized water (60:40) in 8.5 min. The standard costunolide (3) was injected five times and two times for those samples.
The average for the results of each product is given in Table s4. Table s4. Costunolide content of fractions 1, 2, and 3 compared to standard costunolide (3) that showed the costunolide ≥ 97% by HPLC.

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The fractions for No. 5, 7, purified costunolide (1), and standard costunolide (3) were weighed and dissolved in methanol at the final concentration of 0.0808 mg/mL. After that, the mixture was filtered through a 0.45 µm syringe filter and analyzed by HPLC (Agilent 1100 DAD).
The costunolide and was eluted with a mobile phase consisting of methanol and distilled deionized water (60:40) in 22 min. The standard costunolide (3) was injected five times and two times for those samples. The average of the results for each product is given in Table s5.  The fractions No. 5, 7, purified parthenolide (2), and standard parthenolide (4) were weighed and dissolved in methanol at the final concentration of 0.0808 mg/mL. Then the mixture was filtered through a 0.45 µm syringe filter and analyzed by HPLC (Agilent 1100 DAD). The HPLC system consisted of a C-18 reversed-phase column (Vertisep usp 250L 4.6 mm). The parthenolide was eluted with a mobile phase consisting of methanol and distilled deionized water (60:40) in 8.9 min. The standard parthenolide (4) was injected five times and two times for those samples. The average of the results for each product is given in Table s6.   (4)). Asterisk (*) and (**) denote a significant difference from the control groups (P < 0.05 and P < 0.01).
27  (4)). Asterisk (*) and (**) denote a significant difference from the control groups (P < 0.05 and P < 0.01). f) Densitometry was used to quantitate the protein levels as the percentage of vehicle controls (0.02% DMSO alone without the Cos (1) in culture medium). (c, d, g, h) The total cell numbers were measured using the Trypan blue exclusion method. Data used is the mean ± SD from three independent experiments. Asterisk (*) and (**) denotes a significant difference from the control groups (P < 0.05 and P < 0.01).