Supplementary material
In vitro evaluation of the antioxidant and cytotoxic activities of constituents of the mangrove Lumnitzera racemosa Willd.
Nguyen Phuong Thao,1,2 Bui Thi Thuy Luyen,1,2 Chau Ngoc Diep,2 Bui Huu Tai,2
Eun-Ji Kim,3 Hee-Kyoung Kang,3 Sang-Hyun Lee,4 Hae-Dong Jang,4 Nguyen The Cuong,5
Nguyen Van Thanh,2 Nguyen Xuan Cuong,2 Nguyen Hoai Nam,2 Chau Van Minh,2,* Young Ho Kim*,1
1 College of Pharmacy, Chungnam National University, Daejeon 305–764, Republic of Korea
2 Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
3 Department of Pharmacology, School of Medicine, Institute of Medical Sciences, Jeju National University, Jeju 690-756, Republic of Korea
4 Department of Food and Nutrition, Hannam University, Daejeon 305-811, Republic of Korea
5 Institute of Ecology and Biological Resources (IEBR), VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
* To whom correspondence should be addressed:
Tel.: +82-42-821-5933. Fax: +82-42-823-6566. E-mail: (Kim, Y. H.);
Tel.: +84-43-791-7053. Fax: +84-43-791-7054. E-mail: (Minh, C.V.).
List of supporting data
Cotents / PagesGeneral experimental procedures……………………………………………………………. / 3
Extraction and isolation……………………………………………………………………… / 3
Oxygen radical absorbance capacity assay…………………………………………………... / 4
Cell culture…………………………………………………………………………………... / 5
Cell viability assay…………………………………………………………………………… / 5
Morphological analysis of apoptosis by Hoechst 33342 staining……………………………. / 6
Flow cytometric analysis of apoptosis……………………………………………………….. / 6
Western blot analysis………………………………………………………………………... / 6
Statistical analysis……………………………………………………………………………. / 7
Effect of compounds 1 and 14 on the induction of apoptosis………………………………... / 7
Effect of compounds 1 and 14 on the regulation of apoptosis-related proteins……………... / 7
Effect of compounds 1 and 14 on the regulation of ERK 1/2 MAPK and C-myc…………... / 8
General experimental procedures
Optical rotations were determined on a JASCO P-2000 polarimeter (Hachioji, Tokyo, Japan). The IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany). The 1H (600 MHz), 13C NMR (150 MHz) spectra were recorded on a JEOL ECA 600 NMR spectrometer (Billerica, MA, USA), and TMS was used as an internal standard. Column chromatography (CC) was performed on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany), porous polymer gel (Mitsubishi Chemical, Diaion HP-20, 70×180 mm), octadecyl silica (ODS, Cosmosil 140 C18-OPN, Nacalai Tesque), and YMC RP-18 resins (30-50 μm, Fuji Silysia Chemical, Kasugai, Aichi, Japan). Thin layer chromatography (TLC) used pre-coated silica gel 60 F254 (1.05554.0001, Merck, Darmstadt, Germany) and RP-18 F254S plates (1.15685.0001, Merck, Darmstadt, Germany) and compounds were visualized by spraying with aqueous 10% H2SO4 and heating for 3-5 min.
Extraction and isolation
The dried and milled leaves (1.5 kg) of the mangrove L. racemosa Willd. were extracted separately with methanol at room temperature to afford a MeOH residue. The MeOH extract (70.4 g, A) was concentrated in vacuo and partitioned with n-hexane, CH2Cl2, and n-BuOH to afford an n-hexanes (19.2 g, B), a CH2Cl2 extract (17.8 g, C), and an n-BuOH (23.6 g, D). Bioactivity-guided fractionation of the CH2Cl2- and n-BuOH-soluble fractions were carried out using in vitro antioxidant and cytotoxic assays. Their exhibited potent cytotoxicity toward the HL-60 (IC50 < 5.0 μg/mL) and antioxidant activity (values from 7.0 to 12.0 μM TE). The both n-hexane and aqueous-soluble fractions were inactive in the bioassay system used (IC50S > 50 μg/mL).
The CH2Cl2-soluble fraction was chromatographed using vacuum-liquid chromatography over silica gel (230−400 mesh) with a step gradient n-hexane-EtOAc (100/0→0/100), producing 4 pooled fractions (C1 to C4). Subfraction C-4 was chromatographed over silica gel using n-hexane-acetone (8:1, 6:1, v/v) as solvent, and purified by Sephadex LH-20 column (3.5 × 65 cm) chromatography with elution by 10% H2O in MeOH, affording 1 (5.6 mg), 2 (7.2 mg), 9 ( 6.1 mg), and 29 (7.3 mg). Next, subfraction C-3 was chromatographed over silica gel, eluted by n-hexane-EtOAc (10:1, 8:1, v/v), affording 25 (25.4 mg), 26 (27.3 mg), and 33 (5.5 mg). Then, subfractions C-1/C-2 was combined and subjected reversed-phase (RP) flash CC (YMC Gel ODS-A, 60 Å, 400/500 mesh), eluting with a gradient of MeOH–axetone–H2O (10:1:1, 7:1:0.5, v/v), yielding 24 (89.7 mg), 28 (1.2 g), and 30 (32.5 mg).
The n-BuOH-soluble fraction (23.6 g, D) was subjected to silica gel CC (5.5 × 15 cm), eluted by a gradient of CH2Cl2-MeOH (step by step, 800 mL each). Fractions were pooled after TLC analysis and afforded nine combined fractions (D1 to D9). Fractions D-8 and D-9 were found to be active in inhibiting the proliferation of HL-60 cancer cells (IC50 < 3.5 μg/mL), and were combined, and then chromatographed over a silica gel CC (3.5 × 20 cm), eluted by a gradient of CH2Cl2-MeOH (50:1, 40:1, 30:1, v/v, 250 mL each), to yield six subfractions (D-8.1→D-8.6). Subfraction D-8.6 was chromatographed over silica gel, eluted by CH2Cl2-MeOH (3:1), and YMC RP-18 CC using a solvent system of H2O-acetone (5:1, 3.5:1, v/v), affording 31 (9.4 mg), 32 (7.6 mg), 34 (7.6 mg), 35 (11.3 mg), and 36 (14.5 mg). Next, subfraction D-8.5 was chromatographed over silica gel, eluted by CH2Cl2-MeOH (6:1), and purified by Sephadex LH-20 CC, eluted by methanol-H2O (1.5:1) to yield 10 (5.1 mg), 11 (6.3 mg), 12 (4.4 mg), and 13 (7.5 mg). Similarly, sufraction D-8.4 was also chromatographed over silica gel, eluted by CH2Cl2-MeOH (7.5:1), and purified by Sephadex LH-20 CC, eluted by methanol-H2O (1:1), yielding 5 (12.3 m), 6 (8.3 mg), 7 (5.5 mg), and 8 (13.7 mg). Sufraction D-8.3 was chromatographed over silica gel, eluted by CH2Cl2-MeOH (9:1), and then purified with a column containing Sephadex LH-20 CC, using CH2Cl2-MeOH (1:6) for elution, affording 14 (6.5 mg), 15 (5.6 mg), 16 (5.0 mg), 17 (8.2 mg), and 18 ( 7.9 mg). And compound 3 (5.2 mg), 4 (4.2 mg), 19 (13.8 mg) were purified from subfraction D-8.2 following a two-stage separations beginning with a silica gel CC eluted with EtOAc-MeOH (18:1, 15:1, v/v), followed by an YMC CC with MeOH-H2O (2:1, 1:1, v/v). And finally, subfraction D-8.1 was chromatographed over silica gel, eluted by CH2Cl2-MeOH (15:1, 10:1, v/v), and further purified by Sephadex LH-20 chromatography, eluted with a mixture of H2O-MeOH (1:1), yielding 20 (4.5 mg), 21 (5.9 mg), 22 (5.8 mg), 23 (5.0 mg), and 27 (3.5 mg).
Oxygen radical absorbance capacity assay
The ORAC assay, which has been employed extensively in previous antioxidant studies, was carried out using a Tecan GENios multifunctional plate reader (Salzburg, Austria) with fluorescent filters (excitation wavelength: 485 nm, emission filter: 535 nm). In the final assay mixture, fluorescein (40 nM) was used as a target of free radical attack with AAPH (20 mM) as a peroxyl radical generator in the peroxyl radical-scavenging capacity assay. The analyzer was programmed to record fluorescein fluorescence every 2 min after AAPH had been added. All fluorescence measurements were expressed relative to the initial reading. Final values were calculated based on the difference in the area under the fluorescence decay curve between the blank and test samples. All data are expressed as net protection area (net area). Trolox (1.0 μM) was used as the positive control to scavenge peroxyl radicals. It was used as a control standard and prepared fresh daily. The ORAC value is calculated by dividing the area under the sample curve by the area under the trolox curve, with both areas being corrected by subtracting the area under the blank curve. One ORAC unit is assigned as the net area of protection provided by trolox at a final concentration of 1.0 µM. The area under the curve of the sample is compared to the area under the curve for trolox, and the antioxidative value is expressed in micromoles of trolox equivalent per liter.
Cell culture
The HL-60 and Hel-299 cell lines were obtained from the Korea Cell Line Bank (KCLB) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100.0 mg/mL, respectively) at 37°C in a humidified 5% CO2 atmosphere. The exponentially growing cells were used throughout the experiments.
Cell viability assay
The effects of the extract, fractions, as well as the isolated compounds 1–36 on the growth of human cancer cells were determined by measuring metabolic activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. 1987. Evaluation of a retrazolium-based semiautomated colorimetrie assay: Assessment of radiosensitivity. Cancer Res 47: 943-946). Two human cancer cell lines were used. The HL-60 and Hel-299 cell lines were obtained from the KCLB and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100.0 mg/mL, respectively) at 37°C in a humidified 5% CO2 atmosphere. The exponentially growing cells were used throughout the experiments.
The MTT assays were performed as follows: human cancer cell lines (HL-60; 3 × 105 cells/mL, and Hel-299; 1 × 105 cells/mL) were treated for 3 days with 0.01, 0.1, 1.0, 10.0, 50.0, and 100.0 μM of the compounds or 0.01, 0.1, 1.0, 10.0, 50.0, and 100.0 μg/mL of the fraction. After incubation, 0.1 mg (50.0 μL of a 2.0 mg/mL solution) MTT (Sigma, Saint Louis, MO, USA) was added to each well and the cells were then incubated at 37°C for 4 h. The plates were centrifuged at 1000 rpm for 5 min at room temperature and the media was then carefully aspirated. Dimethylsulfoxide (150.0 μL) was then added to each well to dissolve the formazan crystals. The plates were read immediately at 540 nm on a microplate reader (Amersham Pharmacia Biotech., NY, USA). All the experiments were performed three times and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of the compounds compared to the untreated controls. A dose-response curve was generated and the inhibitory concentration of 50% (IC50) was determined for each sample as well as each cell line.
Morphological analysis of apoptosis by Hoechst 33342 staining
The HL-60 (3 × 105 cells/mL) cell was treated with the IC50 values of compounds 1 and 14 for 24 and 48 h. The cell was incubated in a Hoechst 33342 (culture medium at a final concentration of 10.0 μg/mL) staining solution at 37°C for 20 min. The stained cell was observed with an inverted fluorescent microscope equipped with an IX-71 Olympus camera and photographed (magnification × 200).
Flow cytometric analysis of apoptosis
The HL-60 (3 × 105 cells/mL) cell was treated with the IC50 values of compounds 1 and 14 for 24 and 48 h. After treatment, the cell was harvested and washed two times with 0.01 M phosphate buffered saline (PBS; NaCl 0.138 M; KCl-0.0027 M; pH 7.4) and fixed with 70% ethanol at 4°C for 30 min. The fixed cell was washed with cold PBS, incubated with 50.0 μg/mL RNase A at 37°C for 30 min. After incubated, the cell was stained with 20.0 μg/mL propidium iodide (PI; Sigma, MO, USA) in the dark at 37°C for 15 min. The stained cell was analyzed using an FACS caliber flow cytometer (Becton Dickinson, FL, USA).
Western blot analysis
The HL-60 (3 × 105 cells/mL) cell was treated with the IC50 values of compounds 1 and 14 for 24 and 48 h. After treatment, the cell was harvested and washed two times with cold PBS. The cell was lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2.0 mM EDTA, 1.0 mM EGTA, 1.0 mM NaVO3, 10.0 mM NaF, 1.0 mM dithiothreitol, 1.0 mM phenylmethylsulfonylfluoride, 25.0 μg/mL aprotinin, 25.0 μg/mL leupeptin, 1% Nonidet P-40) and kept on ice for 30 min at 4°C. The lysates were centrifuged at 15,000 rpm at 4°C for 15 min. The supernatants were stored at -20°C until use. Protein content was determined by the Bradford assay (Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Ana Biochem 72: 248−254). The same amount of lysates were separated on 8~15% SDS-PAGE gels and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA) by glycine transfer buffer (192 mM glycine, 25 mM Tris-HCl [pH 8.8], and 20% MeOH [v/v]) at 200 mA for 2 h. After blocking with 5% nonfat dried milk, the membrane was incubated with primary antibody against Bcl-2 (1:500), Bax (1:1000), cleaved PARP (1:1000), cleaved caspase-9 (1:1000), cleaved caspase-3 (1:1000), ERK1/2 (1:1000), phospho-ERK1/2 (1:1000), c-Myc (1:1000), and β-actin (1:5000) antibodies and incubated with a secondary HRP antibody (1:5000; Vector Laboratories, Burlingame, VT, USA) at room temperature. The membrane was exposed on X-ray films (AGFA, Belgium), and protein bands were detected using a WEST-ZOL® plus Western Blot Detection System (iNtRON, Gyeonggi-do, Republic of Korea).
Statistical analysis
Data are presented as the means ± SD of at least three independent experiments performed in triplicate. Statistically significant differences were determined by one-way ANOVA followed by Dunnett’s multiple test using GraphPad Prism 6 program (GraphPad Software Inc., San Diego, CA, USA).
Effect of compounds 1 and 14 on the induction of apoptosis
To determine whether compounds 1 and 14 are able to induce apoptosis in HL-60 human cancer cell line, we examined apoptotic characteristics, including cell cycle arrest and nuclear morphological changes. When the cell cycle distribution was analyzed after treatment with compounds 1 and 14 at IC50 levels after 48 h, an increase in sub-G1 hypodiploid cells was observed (Fig. 4A). Because nuclear morphological changes are critical markers of cell apoptosis, we performed Hoechst staining to confirm nuclear morphological changes of apoptosis induced by tested samples. As a result, compounds 1 and 14 induced the production of apoptotic bodies in HL-60 cells (Fig. 4B).
Effect of compounds 1 and 14 on the regulation of apoptosis-related proteins
To determine whether 1 and 14 regulate apoptosis-related proteins in human cancer cell, we examined the expression of anti-apoptotic proteins and pro-apoptotic proteins by Western blot analysis. Treatment with 1 and 14 induced the downregulation of Bcl-2, an anti-apoptotic protein, and the upregulation of Bax, a proapoptotic protein. Compounds 1 and 14, also induced cleavage of procaspase-9, procaspase-3, and poly (ADP-ribose) polymerase (PARP) in a time-dependent manner (Fig. 5). These data indicate that 1 and 14 are able to induce apoptosis in HL-60 cell through regulation of apoptosis-related proteins.