Supplementary Material s92

SUPPLEMENTARY MATERIAL

Synergistic action of compounds isolated from hexane extract of Ardisia crispa root against tumour promoting effect, in vitro

Looi Ting Yeong1, Roslida Abdul Hamid1*, Latifah Saiful Yazan1, Huzwah Khaza’ai1 and Dayang Erna Zulaikha Awang Hamsin1,2

1Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Malaysia

2Department of Paraclinical Science, Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak, 93150 Kuching, Malaysia

*Corresponding author Phone: +603 8947 2341 Fax: +603 8943 6178 E-mail: [email protected]

Abstract:

Isomeric mixture of α,β-amyrin (triterpene) and 2-methoxy-6-undecyl-1,4- benzoquinone (quinone) isolated from Ardisia crispa root hexane (ACRH) extract were reported with anti-inflammatory properties in vivo. Considering the close association between inflammation and cancer, on top of the lack of antitumor study on those compounds, this study aimed to determine the potential of both compounds against tumour promotion in vitro, either as single agent or in combination. Triterpene and quinone compounds, as well as triterpene-quinone fraction (TQF) and ACRH were subjected to inhibition of Epstein-Barr virus-early antigen (EBV-EA) activation assay for that purpose. With comparison to curcumin (positive control), inhibition against EBV-EA activation occurred in the order: ACRH > TQF ≥ curcumin > α,β-amyrin ≥ 2-methoxy-6-undecyl-1,4-benzoquinone. These findings reported, for the first time, antitumor promoting effect of α,β-amyrin and 2-methoxy-6-undecyl-1,4-benzoquinone from the roots of Ardisia crispa, which was enhanced when both compounds act in synergy.

Keywords: Ardisia crispa – inflammation - antitumor promoting – inhibition of EBV-EA activation assay

3. Experimental 3.1. Chemicals Silica gel (70-230 mesh) and thin-layer chromatography (TLC) plate (precoated silica gel 60 F254) for compound isolation were purchased from Merck (Darmstadt, Germany). All reagents used were of analytical grade available commercially. 3.2. Plant material Roots of Ardisia crispa were collected from Machang, Kelantan, Malaysia in April 2010 and deposited (voucher specimen no.: 20841) in the herbarium of Universiti Kebangsaan Malaysia, Selangor, Malaysia upon identification by Dr. Roslida Abdul Hamid. 3.3. Extraction and isolation Protocols for plant extraction and compound isolation were carried out according to method described by Roslida (2004). Ardisia crispa roots were dried at 40°C for 3 days and grinded into fine powder by using laboratory mill (Retsch, Germany). Ground roots (200 g) were extracted with 80% aqueous ethanol in the proportion of 1:15 (w/v) using Soxhlet apparatus (80-90°C) for 24 hours. The extract was concentrated in a rotary evaporator at 40°C, under reduced pressure, to give crude aqueous ethanolic (ACRE; 129.5 g, 6.5%) extract. This was followed by subsequent fractionation with n-hexane (3 x 500 ml, 48 hours each) which was then filtered and concentrated under reduced pressure. Ardisia crispa root hexane (ACRH; 35.3 g, 27.4%) extract was obtained after the concentrates dried at room temperature. Chromatography of ACRH (5 g) over silica gel-packed column (200 g, 70-230 mesh, 75 cm x 5 cm, flow rate 1 ml/min) eluted with n-hexane:ethyl acetate (v/v) (9:1, 8:2, 7:3, 6:4; 500 ml each) furnished 99 fractions (frs.): 1-25 (9:1); 26-48 (8:2); 49-70 (7:3); 71-99 (6:4) (Figure S3). TLC, with chloroform as developing solvent, was performed to detect the presence of compounds of interest in the eluates (collected at 20 mls interval). Retention factor (Rf) obtained was compared to that of the reference standards kindly provided by Dr. Roslida Abdul Hamid: AC1 [α,β-amyrin (triterpene); > 97% by GC] and AC2 [2-methoxy-6-undecyl-1,4-benzoquinone (quinone); ~65% by GC]. Compounds of interest were found abundant in frs. 41-99. Rechromatography of pooled fractions (frs. 41-99) further yielded frs. 1-24 (8:2); 25-47 (7:3); 48-75 (6:4). Repeated rinsing of frs. 22-31 with n-hexane afforded compound 1, frs. 45-65 afforded compound 2 and frs. 32-44 gave triterpene-quinone fraction (TQF). 3.4. Confirmation of compounds isolated by GC-MS The system consisted of an Agilent model 5973 MSD gas chromatograph equipped with a HP-5 MS column (30 m x 0.25 mm x 0.25 µm). 1 µl of isolated compound or reference standard (AC1 and AC2) was injected in splitless mode at an injector temperature of 250°C. Helium, at a flow rate of (1 ml/minute), was used as the carrier gas. The oven temperature was programmed from 70°C with gradual increase to 300°C in 6 minutes and was held for 29 minutes. The mass spectrometer was electron impact ionization mode. Library mass spectra search was performed via National Institute of Standard and Technology (NIST) library with comparison to data of reference standards obtained from the present analysis, as well as from the literature. 3.5. Cell line and chemicals Raji (human B-lymphoblastoid) cell line was provided by Dr. Lim Yang Mooi (Department of Pre-clinical Sciences, Universiti Tunku Abdul Rahman); whilst EBV-EA positive NPC serum was obtained as generous gift from Dr. Paul Lim Vey Hong (Western Medical Division, Tung Shin Hospital) with ethical approval from the University Research Ethics Committee (JKEUPM) [UPM/FPSK/100-9/2-JKEUPM (JSB_Mac(12)15)]. Raji cells were maintained in medium RPMI 1640 supplemented with L-glutamine (0.2 g/l), foetal bovine serum (10%), streptomycin (100

µg/ml) and penicillin (100 IU/ml), in a humidified atmosphere of 5% carbon dioxide (CO2) at 37°C. 3.6. Determination of cytotoxicity Cytotoxicity was evaluated using the tetrazolim (MTT) based colorimetric assay according to method described by Hsu et al. (2005), with slight modification. Various concentrations (50, 10, 2, 0.4 and 0.08 μg/ml) of samples (ACRH, TQF, compound 1 and 2, and curcumin) were prepared in dimethylsulfoxide (DMSO). Raji cells (1 x 106 cells/ml) were seeded in a 24-well flat-bottomed tissue culture plate and treated with 0.08-50 μg/ml of samples. The plate was incubated at 37ºC for 48 hours. Then, 50 μl of treated cell suspension was transferred into 96- well flat bottomed tissue culture plate, followed by addition of 10 μl of MTT solution [5 mg/ml in phosphate buffered saline (PBS)]. The plate was incubated at 37°C for another 4 hours and finally, 150 μl of DMSO solution was added into each well. Absorbance was measured at 570 nm with a reference wavelength of 630 nm. The concentration that gave 50% inhibition of cell viability (IC50) when compared to control was determined. 3.7. Inhibition of EBV-EA activation assay The indicator cells (Raji cells, 1 x 106/ml) were incubated at 37°C for 48 hours in 1 ml of medium containing n-butyric acid (4 mmol), TPA (32 pmol, 20 ng in DMSO, 2 µl) as inducer and various amounts of samples (0.008-5 µg/ml) in 5 µl DMSO (Ito et al. 1999). After 48 hours, smears were made from the cell suspension and activated cells that were stained by EBV-EA positive serum were detected by an indirect immunofluorescence technique (Henle & Henle 1966). Triplicate assays were carried out where at least 500 cells were counted for each measurement, and the number of stained cells (positive cells) was recorded. The average EBV- EA induction of the sample was expressed as a relative ratio to the control experiment (100%) which was carried out only with n-butylic acid (4 mmol) plus TPA (32 pmol). The inhibitory rate (IR) of each test sample against the EBV activation was classified into four ranks as follows: ++ + (strongly active; IR ≥ 70%); ++ (moderately active; 70% > IR ≥ 50%); + (weakly active; 50% > IR ≥ 30%); - (inactive, 30% > IR) (Murakami et al. 2000). 3.8. Statistical analysis Data were expressed as mean ± standard error of mean (S.E.M.). Statistical analysis was carried out with SPSS 20.0 software by using one-way analysis of variance (ANOVA) followed by Least Significant Difference (LSD) post hoc test to compare means between groups. Values with P<0.05 were considered statistically significant. Table S1. Major ion peaks of α,β-amyrin from MS of compound 1 and AC1. α-amyrin (m/z, relative intensity %) β-amyrin (m/z, relative intensity %) Compound 1 AC1* Literature+ Compound 2 AC1* Literature+ 426 (8) [M+] 426 (5) [M+] 426 (8) [M+] 426 (5) [M+] 426 (5) [M+] 426 (7) [M+] 411 (2) 411 (2) 411 (8) 411 (2) 411 (2) 411 (17) 393 (13) 393 (3) 218 (100) 218 (100) 218 (100) 218 (100) 218 (100) 218 (100) 203 (20) 203 (20) 203 (21) 203 (45) 203 (44) 203 (52) 189 (17) 189 (18) 189 (30) 189 (14) 189 (14) 189 (16) * Reference standard (AC1) provided by Dr. Roslida Abdul Hamid. + Refers to patent publication by Nawar (2010). Table S2. Major ion peaks of 2-methoxy-6-undecyl-1,4-benzoquinone from MS of compound 2 and AC2. Compound 2 (m/z, rel. int., %) AC2 (m/z, rel. int., %)* 292 (61) [M+] 292 (58) [M+] 193 (13) 193 (12) 179 (25) 179 (24) 166 (13) 166 (12) 154 (100) 154 (100) 139 (12) 139 (13) 124 (15) 124 (14) 109 (12) 109 (12) 95 (7) 95 (6) 81 (7) 81 (7) 69 (26) 69 (27) 55 (17) 55 (17) * Reference standard (AC2) provided by Dr. Roslida Abdul Hamid. Figure S1. Chemical structure of (a) α-amyrin; (b) β-amyrin. Figure S2. Chemical structure of 2-methoxy-6-undecyl-1,4-benzoquinone Figure S3. Schematic diagram of extraction and isolation from Ardisia crispa roots to obtain compound 1, compound 2 and TQF via column chromatography and TLC.

Figures Figure S1

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