RESEARCH ARTICLE Anti-Proliferative Activity and Apoptosis Induction of an Ethanolic Extract of Boesenbergia pandurata (Roxb.) Schlecht. against HeLa and Vero Cell Lines

Rhizomes of Boesenbergia pandurata (Roxb.) Schlecht have been reported to contain active compounds with anticancer properties. This research was carried out to examine anti-proliferative and apoptotic induction against HeLa and Vero cells-line. Dried powder of B. pandurata rhizomes was extracted by a maceration method using 90% ethanol. Cytotoxic assays to determine IC 50 and anti-proliferative effects were carried out by MTT methods. Observation of apoptosis was achieved with double staining using acridine orange and ethidium bromide. The results showed that ethanolic extract of B. pandurata was more cytotoxic against HeLa cells (IC 50 of 60 µg/ mL) than Vero cells (IC 50 of 125 µg/mL). The extract had higher anti-proliferative activity as well as apoptotic induction in HeLa than Vero cells. Therefore, it was concluded that the ethanolic extract of B. pandurata had anti-proliferative as well as apoptosis induction activity dependent on the cell type.


Introduction
Natural products may serve as a source of many phytochemicals that possess various bioactivities including to prevent cancer, cardiovascular disease, infection and inflammation (Rabeta et al., 2013;Palasap et al., 2014). Therefore, it is promising to discover anticancer compounds from natural product. Boesenbergia pandurata (Roxb.) Schlecht.), known as temu kunci in Indonesia and krachai in Thailand, is one of the edible plants on the ginger family (zingiberaceae) that distributed in Southeastern Asian such as Indonesia, Thailand, and Malaysia. The rhizome has been used tradisionaly for a spice and folk medicine such as treatment of inflammation and gastrointestinal disorder (Kementrian Kesehatan Republik Indonesia, 2010) Several studies on the rhizomes extract of B. pandurata and their isolated compounds have showed some pharmacological activities. The ethanol extracts of this rhizome showed high cytotoxic activity against the Hep2 cell-lines (Kamkaen et al., 2006), and the methanol extract showed an anti-proliferative effect against ovarian (CaOV3), breast (MDA-MB-231 and MCF-7), cervical (HeLa) and colon (HT-29) cancer celllines (Jing et al., 2010 (Yun et al., 2006;Ching et al., 2007;Tanjung et al., 2013). Boesenbergin A isolated from Boesenbergia rotunda, a chalcone has been found as anti-cancer effect through the induction of apoptosis against human non-small cell lung cancer (A549 cells) (Isa, et al., 2013). Panduratin A, a cyclohexenyl chalcone derivative, showed antiproliferative effect and induced apoptosis on human breast cancer (MCF-7) and human colon adenocarcinoma (HT-29) cell-lines (Kirana et al., 2007). In addition, panduratin A also found to inhibit the angiogenesis process at in-vitro and in-vivo levels (Lai, et al., 2012). Hydroxypanduratin A and panduratin A showed anti-inflamatory activity on the TPA-induced ear edema assays in rats (Tuchinda et al., 2002) and murine macrophage-like RAW 264.7 cells-line through inhibition of PGE2 and TNF-α release (Tewtrakul et al., 2009). Both compounds also showed good competitive inhibitory activities towards dengue 2 virus NS3 protease (Kiat et al, 2006). Major antioksidan activity was also showed on hydroxypanduratin A and panduratin A, through the inhibitory activities of rat brain lipid peroxidation (Shindo et al., 2006). Pinocembrin and pinostrobin, flavanones isolated Kaemferia pandurata showed antioxidant and cytotoxic activity (Tanjung et al., 2013).
All the activity of these substances isolated from B. pandurata, antioxidant, anti-inflammation, antimutagenic, and anti-angiogenesis (Shindo et al., 2006;Kirana et al., 2007;Morikawa et al., 2008;Tewtrakul et al., 2009) are associated with chemoprevention activity. Therefore, it is needed to investigate the antiproliferative and apoptosis effects of the rhizome extract of B. pandurata grown at Yogyakarta against HeLa and Vero cells.

Plant material
Rhizomes of B. pandurata were collected from Kalibawang, Kulonprogo, Yogyakarta, Indonesia, and was determinated by Pharmaceutical Biology Department, Faculty of Pharmacy, Universitas Gadjah Mada. This rhizome was sliced and dried at 50 o C then grounded to a fine powder. Extract was prepared by maceration method using 90% ethanol and then evaporated Semisolid extract was quantified by TLC scanner using pinostrobin as an active marker. This extract was reffered as EEBP.

Cytotoxic assay
The cytotoxicity was determined by MTT assay. HeLa and Vero cells at density of 10 5 cells/mL were distributed into 96 wells of plates, treated with various concentration of B. pandurata extracts dissolved in dimethylsulphoxide (DMSO 1,952 -1000 µg/mL), followed by incubation for 24 hr. Each well was added by 15 µl 0.3% MTT in PBS, and after 6 h the reaction was stopped by 10% SDS. The formation of purple phormazan crystal was identified by ELISA reader at 550nm. The IC 50 was determinate, as concentration of the extraxt required that inhibit of 50% cells growth (Doyle and Griffiths, 2000).

Anti-proliferative assay
HeLa and Vero cell (2x10 4 )/mL was grown and starvated in culture media (DMEM) with 0.5% FBS for 24hr in 96 wells plate, and added with B. pandurata extract at a concentration of 7.5, 15, and 30ug/ml in DMSO. The viability of cells was assessed by MTT assay. Population of viable cells in each group treatment was evaluated at 24, 48, and 72h.

Induction of apoptosis assay
Cells (5x10 4 )/well were grown in cover slip at 24 wells plates, treated with B. pandurata extract at a dose of IC 50 and incubated for 24hr. The cell was rinsed by PBS and added 10µL ethidium bromide-acridine orange (5µg/mL in PBS/well). The cells were evaluated by fluorescent microscope 400X. The apoptosis was defined as the appearance of apoptotic bodies, cytoplasmic changes, and chromatin condensation. The dead cells were showed at orange color, and survived cells were green.

Statistical analysis
Absorbance value from cytotoxic and inhibition of proliferation converse to viability cell (%).
% %viable cells = absorbance of cells-absorbance of medium control absorbance of cells medium control -absorbance of medium control x100 Numerical data for treatment cells was subjected to ANOVA analysis. Significance of differences among treatment on the cells was determined by Tukey's test used SPSS statistical package (version 17.0). The p<0.05 were considered as statistically significant.

Cytotoxic activity of ethanolic extract of B. pandurata rhizomes
Cytotoxic test was done to find out the potency of   Anti-proliferative assay rhizome B. pandurata extract against HeLa and Vero cell-lines was done by doublingtime assay, and used concentration around IC 50 result, that were 0; 7,5; 15; and 30 µg/mL. The result showed that the extract could stronger inhibits the proliferation of HeLa cell at 24, 48, and 72 hours (Figure 2A), than Vero cells ( Figure 2B). Increasing extract concentration could increase the inhibition of cells proliferation. The extracts could inhibit HeLa cells proliferation from 14% to 87%. However, at the concentration of 30µg/mL, the extract could only inhibited 28% proliferation of Vero cells.

Induction of apoptosis
Apoptosis assay was done by double staining using ethidium bromide-orange acridine. It was demonstrated that at the extract concentration of 30 µg/mL caused the formation of apoptotic body as shown by the orange reddish cells on HeLa cells, this indicating the cells were undergo apoptotic process ( Figure 3A). The orange reddish of the cells is caused by membrane permeability damage, so that, ethidium bromide penetrate into cells (Meiyanto et al., 2008). While in Vero cells the formation of apoptotic body was very low even at the concentration of 30 µg/mL, indicating that apoptotic process was very low ( Figure 3B). Incubation with EEBP significantly increased apoptotic HeLa cells were 28% than without EEBP. While in vero cells line, treatment EEBP didn't show significantly different with control (Tabel 1)

Discussion
The use natural products now have given of exceptional value in the control of cancer. Many chemoprevention and anti-cancer agents currently used have origin in plants (Al-oqail et al., 2013;Hussain et al., 2015). Using natural products from plants or herbs have some andvantages, having low side effect and low cost and also being easily accessible in comparison to common treatment methods, may play an important role in the treatment of the cancer (Fattahi et al., 2013) One of the plants that potentially as chemopreventife and anti-cancer agent is B. pandurata. Many studies of this edible plant showed many bioactivity including cytotoxic and apoptotic induce against cancer cells line (Kirana et al., 2006;Jing et al., 2010) In this study, extraction of B. pandurata rhizomes using maseration method with 90% ethanol produced semisolid extract with pinostrobin content of 0.49%. The cytotoxic activity assay of ethanolic extract of B. pandurata (EEBP) against HeLa cells line resulted IC 50 of 56 µg/mL.This result supported the previous study of  finger root (Boesenbergia rotunda syn. B. pandurata) collected from Malaysia, which had the cytotoxic activity against HeLa cells line, with the IC 50 of 65.50 µg/mL (Jing et al., 2010) and from Tawangmangu Indonesia was 87 µg/mL (Handoko et al., 2011). Differences of origin locally and extraction method of rhizomes produce different quality, quantity, and activity of extract. Many compounds also showed citotoxic activity against HeLa cells-line, such as phytophenolic from Caesalpinia mimosoides (Palasap et al., 2014) and Aloe vera crude extract (IC 50 =60%) (Hussain et al., 2015). Compounds with the IC 50 value less than 100 µg/mL has the potency as a cancer chemoprevention (Meiyanto, et al., 2008). Result of this study, indicated that EEBP more active on inhibiting proliferation of HeLa than Vero cells-lines, so that the extract could be used as a potent candidate for cancer chemoprevention agent. This finding related to the result of study which reported that finger root (B. rotunda) methanolic extract could inhibit proliferation of HeLa cells but not on normal cells (fibroblast cells -lines 3T3) (Jing et al. 2010). Many phytochemicals also demonstrated inhibited proliferation of HeLa cells line such as ethanol extract of bark and leaves of Erythrina fusca Lour (Meiyanto et al., 2003) and Sauromatum giganteum showed inhibition rates 6,24% (Gao et al., 2014).
The induction of apoptosis is a hallmark of cancer treatment (Hanahan and Weinberg, 2011). Induce of cancer cells to apoptosis is one of target for cancer chemoprevention, since no inflammation is found during this process. The induction of apoptotic effect of EEBP against of HeLa cells-line was supported the previous finding on HT-9 human colon adenocarcinoma cells line treated with panduratin A isolated from B. pandurata rhizome by inhibition of procaspase 9, 8, 3, 6 proteins, cause apoptotic process (Yun et al., 2006). Previouse study showed that a large spectrum of chalcone derivatives with proapoptotic properties have been found in various edible or medical plants (Orlikova et al., 2011). Beside panduratin A other coumpounds, chalcone, and cardomomin, enhanced TRAIL-induced apoptosis in TRAIL-resistant DLD1 cells-line (colorectal adenocarcinoma), increased expression of death receptor (DR4 and DR5) and reduced Bcl-xL levels following cardamomin treatment of cells (Hsu et al., 2006). The apoptotic induction effect of EEBP found to be selective to cancer cells show that it could be a potent cancer chemoprevention agent.
Ethanolic extract of B. pandurata had more potent cytotoxic activity, inhibition of proliferation, and apoptotic induction on HeLa cells-line compare to Vero cellsline. The extracts could be as candidate agent of cancer chemoprevention.