Synergistic action of compounds isolated from the hexane extract of <i>Ardisia crispa</i> root against tumour-promoting effect, <i>in vitro</i>

<div><p>An isomeric mixture of α,β-amyrin (triterpene) and 2-methoxy-6-undecyl-1,4-benzoquinone (quinone) isolated from the <i>Ardisia crispa</i> root hexane (ACRH) extract was reported to possess anti-inflammatory properties <i>in vivo.</i> Considering the close association between inflammation and cancer, on top of the lack of antitumour study on those compounds, this study aimed to determine the potential of both compounds against tumour promotion <i>in vitro</i>, 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. Compared with 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, the antitumor-promoting effect of α,β-amyrin and 2-methoxy-6-undecyl-1,4-benzoquinone from the roots of <i>A. crispa</i>, which was enhanced when both compounds act in synergy.</p></div

Antioxidant properties of ginger (<i>Kaempferia angustifolia</i> Rosc.) and its chemical markers

<p>The nutritional value of the rhizomes of <i>Kaempferia angustifolia</i> was measured through the antioxidant properties of various extracts and the determination of the bioactive compounds. The chloroform and methanol extracts of the rhizomes of <i>Kaempferia angustifolia</i> showed strong free radical scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with values of 615.92 mg Trolox equivalent (TE)/g each. The methanol extract also exhibited the strongest antioxidant properties in the azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assay with a value of 38.87 mg TE/g. The hexane extract gave the strongest reducing ability in cupric reducing antioxidant capacity (CUPRAC) assay with a value of 901.76 mg TE/g, whilst the ethyl acetate extract exhibited the strongest reducing ability against ferric reducing antioxidant power (FRAP) with a value of 342.23 mg TE/g. Column chromatographic separation on the rhizomes of <i>Kaempferia angustifolia</i> afforded boesenboxide (<b>1</b>), crotepoxide (<b>2</b>), 2ˊ-hydroxy-4,4ˊ,6ˊ-trimethoxychalcone (<b>3</b>), kaempfolienol (<b>4</b>), and zeylenol (<b>5</b>), which were elucidated by spectroscopic methods. Kaempfolienol (<b>4</b>) was the strongest free radical agent against DPPH with a value of 443.92 mg TE/g, whilst 2ˊ-hydroxy-4,4ˊ,6ˊ-trimethoxychalcone (<b>3</b>) exhibited the strongest antioxidant properties with values of 42.23, 1497.22, and 781.53 mg TE/g against ABTS, CUPRAC, and FRAP assays, respectively.</p

Extraction and isolation of ethyl acetate extract of <i>Dillenia suffruticosa</i>.

<p>Sequential solvent extraction using solvents with increasing polarity (hexane< dichloromethaneD. <i>suffruticosa</i>. The extract obtained was subjected to isolation using column chromatography and thin layer chromatography by using different solvent systems.</p

Expression level of the apoptotic-related genes in MCF-7 cells treated with EADs as determined by GeXP analysis.

<p>EADs upregulated the expression of <i>Bax</i> and <i>p21</i> and downregulated the expression of <i>Bcl-2</i> and <i>caspase-9</i>. The expression of genes was normalized against beta actin and compared to the control. The data are represented as relative expression of genes in bars±SD of at least three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p

List of genes with their respective primers for GeXP multiplex analysis.

<p>Forward universal primer sequence (5’-AGGTGACACTATAGAATA-3’); Reverse universal primer sequence (3’-GTACGACTCACTATAGGG-5’).</p><p>List of genes with their respective primers for GeXP multiplex analysis.</p

Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.

<p>(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk ^a indicates statistically significantly different from the untreated control (P<0.05).</p

Involvement of caspase in EADs-induced apoptosis in MCF-7 cells.

<p>General inhibitor Z-VAD-FMK did not inhibit the induction of apoptosis by EADs suggesting it is caspase-independent. (A) represents mean percentage of three independent experiments±SD. (B) Comparison of the percentage of apoptotic cells between caspase inhibitor negative group and caspase inhibitor positive group at different concentrations. The data are presented as mean±standard deviation of three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p

Proposed signalling pathway of EADs-induced apoptosis in MCF-7 cells.

<p>It is postulated that EADs induces apoptosis in MCF-7 cells via the production of oxidative stress, p53- and p21-dependent cell cycle arrest, activation of JNK and NF-κB pathways and inactivation of AKT and ERK pathways. Regulation of these pathways eventually leads to the execution of mitochondrial-dependent and caspase-independent apoptosis.</p

Chemical structure of the compounds isolated from EADs.

<p>Structures of the isolated compounds were elucidated using 1^H and 13^C NMR spectroscopy. The isolated compounds were identified as kaempferide (<b>1</b>), kaempferol (<b>2</b>), protocatechuic acid (<b>3</b>), gallic acid (<b>4</b>), 3-epimaslinic acid (<b>5</b>) and β-sitosterol-3-O-β-D-glucopyranoside (<b>6</b>).</p

Involvement of oxidative stress in EADs-induced apoptosis in MCF-7 cells.

<p>(A) and (B) represent the percentage of viability of MCF-7 cells pre-treated with vitamin C, α-tocopherol or EADs alone, at 24 and 48 hours, respectively. (C) Level of ROS in MCF-7 cells as determined using DCFH-DA assay. Data showed that pre-treatment of MCF-7 cells with α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of EADs (P<0.05). On the other hand, EADs attenuated the intracellular ROS in MCF-7 cells in a concentration-dependent manner (P<0.05). The data are presented as mean±standard deviation of three replicates from at least three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).</p