EVALUATION OF CYTOTOXICITY OF LEAF AND RHIZOME EXTRACTS OF Alpinia calcarata

The cytotoxicty of Alpinia calcarata rhizome and leaf extracts, fractions and essential oils were evaluated in vitro against human lung NCI-H460 and cervical HeLa cancer cell lines using sulphorhodamine-B assay. Although an array of bioactivities of A. calcarata rhizome have been reported previously, no attempt has been made to study the cytotoxicity of rhizome in human lung NCI-H460 and cervical HeLa cancer cell lines. In the present study, both the leaf and rhizome extracts (ethanolic and water) along with their fractions (hexane, dichloromethane, ethylacetate, butanol and water) and essential oils against human lung (NCI-H460) and human cervical (HeLa) cancer cell lines were investigated. Fresh rhizomes and leaves of Alpinia calcarata, collected from Western Province of Sri Lanka were used to obtain the extracts. The essential oils were obtained by hydro-distillation. All the samples were stored at 4 °C. The extracts, fractions and essential oils demonstrated a varying degree of growth inhibition against NCI-H460 cell line. Several fractions showed high growth inhibitory activity (4-94%). The growth inhibition order was, rhizome water extract (107.7%) < rhizome ethanolic extract (68.2%) < leaf ethanolic extract (-25.1%) < leaf water extract (-72.3%) < rhizome oil (93.0%) < leaf oil (-94.3%). Dichloromethane fraction of leaf ethanolic extract (-33.8%) showed the most promising inhibition at GI50: 30.6 μg/mL on NCI-H460. For HeLa cells, the ethyl acetate (-0.5%), butanol (-5.0%) and aqueous fractions (-18.1%) of rhizome water extract showed high growth inhibitions. The potency of the growth inhibition was, rhizome ethanolic extract (76.56%) < rhizome water extract (16.65%) < leaf oil (15.88%) < rhizome oil (-49.34%). Efficacy and mechanisms of action in various normal and cancer cell models coupled with bioassay-guided purification to identify active anticancer compound(s) from the crude extract will be useful.


Introduction
Cancer is a complex group of diseases incorporating physical, environmental, metabolic, chemical and genetic factors.It is one of the leading causes of morbidity and mortality worldwide, accounting for 14 million new cases and over 8 million deaths in 2012.The number of new cases of cancer is expected to reach to 22 million in two decades (WHO, 2014).Due to adverse effects, increasing resistance and unaffordable prices of cancer chemotherapy agents, it is necessary to discover non toxic potential drugs from natural resources for the treatment and prevention of cancer (Demainet al., 2011).
Two bis-labdanic diterpenoids, named calcaratarins D & E, isolated from the rhizome of Alpinia calcarata grown in China, were found to have cytotoxic activity against human KB cells (Ling-Yi LY Kong et al., 2002).The ethanol extract of rhizome at 8 mg/kg/day significantly reduced Ehrlich ascites carcinoma in Swiss Albino mice (Perveen et al., 2012).
n-Hexane and dichloromethane extracts of leaves and rhizomes of A. scabra grown in Malaysia were screened for cytotoxic effect against SKOV-3 and MCF-7 cells.The n-hexane and dichloromethane extracts of rhizome showed a high cytotoxic effect against both cells with IC50 values of 8.3 and 7.0 μg/mL, respectively (Ibrahim et al., 2010).Although an array of bioactivities of A. calcarata rhizome have been reported previously (Arambewela et al., 2004), no attempt has been made to study the cytotoxic activity of the rhizome and leaf in human lung NCI-H460 and cervical HeLa cancer cell lines.Additionally, in previous studies, only the rhizome extracts were studied.However, in present study, A. calcarata rhizome and leaf extracts (aqueous and ethanolic) along with their fractions (hexane, dichloromethane, ethyl acetate, butanol and water) and essential oils against human lung (NCI-H460) and human cervical (HeLa) cancer cell lines were investigated.

Plant collection
A. calcarata rhizomes and leaves were collected from a home garden in Western Province, Sri Lanka, during July 2011.The plant was identified by comparing with an authenticated sample that was grown in Industrial Technology Institute and deposited in Royal Botanical Gardens, Peradeniya, Sri Lanka (Voucher specimen number AC 01).

Chemicals and cancer cell lines
Human lung NCI-H460 and human cervical HeLa cancer cell lines were obtained from the National Cancer Institute, USA.FBS was purchased from Gibco and sulforhodamine-B from MP Biomedicals, USA.All the other chemicals were purchased from Sigma, USA.

Extraction, fractionation and distillation of oil
The collected plant material was washed under running water, cut into small pieces and shade dried for 10 days.The dried samples were extracted with water (refluxing for 5 h) and 70% ethanol (Soxhlet extraction for 6 h) separately.The water extract was evaporated under reduced pressure (150 mbar at <50 °C) in a rotary evaporator, and freeze dried.The dried crude extracts were fractionated with hexane, dichloromethane, ethyl acetate, butanol and water.For the extraction of oils, dried rhizomes and leaves (500 g each) were subjected to hydro-distillation using Clevenger apparatus for 6 h.The oils were stored at 4 °C in air-tight containers until used for the cytotoxicity test.

Cytotoxic activity test
The cytotoxic activity was evaluated by sulphorhodamine-B assay (Monk et al., 1991).Alpinia calcarata extracts, oils and fractions, were evaluated against human lung NCI-H460 and human cervical HeLa cancer cell lines.Cells were plated in 96microwell plates (NCI-H460: 7500 cells/well; HeLa: 10,000 cells/well) and incubated at 37 °C for 24 h in a humidified 5% CO2 incubator.Doxorubicin, an anticancer drug was used as the standard.Stock solutions of ethanolic and water extracts of A. calcarata (40 mg/mL), fractions (20 mg/mL) and positive control doxorubicin (1 µM) were prepared in DMSO.For the initial screening, 100 μL of ethanolic and water extracts and the oils derived from rhizomes, and leaves (250 μg/mL) and the fractions (100 μg/mL) were added into each well and incubated.Fifty microliter of 50% cold trichloroacetic acid (TCA) was added in the wells and left for 30 min at room temperature.The wells were then washed with distilled water and dried overnight.Sulforhodamine-B (SRB) solution (100 L, 0.4% wt/vol in 1% acetic acid) was added.After 30 min, the unbound SRB was removed by washing with 1% acetic acid, and air-dried at room temperature.The protein bound stain was solubilized with 10 mM Tris-base (pH 10.2), the plates were shaken for 5 min by using a plate shaker and the absorbance was measured at 515 nm using SpectraMax Plus 384 microplate reader.The absorbance of the appropriate blanks, including sample blanks, and control (without plant sample), was used to calculate the percentage net growth (%NG), and the cytotoxicity of the extracts and fractions.A. calcarata extracts and the fractions were further evaluated at various concentrations (extracts: 15.62, 31.25,62.5, 125 and 250 μg/mL; fractions: 6.25, 12.5, 25, 50 and 100 µg/mL) to study the dose response.The growth inhibition and cytotoxicity of extracts and fractions was represented as GI50 (μg/mL) values.The standard drug doxorubicin demonstrated the GI50 (0.07 ± 0.00 µg/mL) and LC50 (0.86 ± 0.00 µg/mL).All cytotoxic activity was assessed at 12 h following the treatment.The sulforhodamine-B assay was used to quantify the cytotoxic effects.

Gas chromatography-Mass spectrometric analysis
Oil samples (rhizome oil, leaf oil and the hexane fraction of leaf ethanolic extract: 0.5 L each) were injected into an Agilent 7890A/7000AGC triple quadrupole mass spectrometer system (GC/QQQ) for identification of compounds using 1:60 split injection ratio.A HP-5MS fused silica capillary column of 30 m length, 0.25 mm inner diameter and 0.25 µm film thickness (J&W Scientific, USA) was used.
The initial oven temperature was 40 C for 5 min ramped to 280 C at 10 C /min, and held there for 5 min.The linear velocity of the carrier gas (Helium) was constant at 40 cm/s.The inlet temperature was 250 C, transfer line temperature was 250 C and EI ionization source temperature was 230 C.Mass spectra were acquired using full scan monitoring mode with a mass range of 50-500 m/z.Data Analysis was done by using Agilent Mass Hunter software version B.04.00 and National Institute of Standards and Technology (NIST) Data base Library 2010.

Statistical analysis
Results are presented as Mean±Standard Error.Significance of differences between groups was assessed with use of one way ANOVA.

Results
Hexane, dichloromethane, ethylacetate, butanol and aqueous fractions of A. calcarata ethanolic and water extracts as well as the oils derived from rhizomes and leaves demonstrated a varying level of growth inhibition (%NG) against lung cancer cell line NCI-H460 (Figure 1, Table 1 and 2).
The butanol fraction from both rhizome ethanolic extract and rhizome water extract, dichloromethane and ethylacetate fractions of the leaf ethanolic extract, and the hexane, dichloromethane, ethylacetate, butanol and aqueous fractions of A. calcarata ethanolic and water extracts, as well as the oils derived from rhizomes and leaves demonstrated a varying level of growth inhibition (%NG) against lung cancer cell line NCI-H460 (Fig. 1, Table1 and 2).The butanol fraction from both rhizome ethanolic extract and rhizome water extract, dichloromethane and ethylacetate fractions of the leaf ethanolic extract and the hexane and dichloromethane fractions of the leaf water extract showed a higher inhibitory activity ranging between 4-94%.Results revealed differences among the extracts and fractions for growth inhibition.Leaf ethanolic extract showed a high growth inhibition as compared to the leaf water extract and the oils.
For the HeLa cell line, the ethylacetate and aqueous fractions of rhizome ethanolic extract showed significantly moderate growth inhibition while ethylacetate, butanol and aqueous fractions of rhizome water extract showed a high growth inhibition (Table 3).Rhizome oil showed highly potent growth inhibition on HeLa cells as compared to leaf oil.Among the extracts and oils tested, the potency of the growth inhibition was ranked as rhizome ethanolic extract < rhizome water extract < leaf oil < rhizome oil (Table 3).

Discussion
The major aim of this study was to identify potential anticancer extracts that were effective, even at low doses.In order to achieve this aim, the maximum test concentration was set at 100 μg/mL as the criteria for identifying extracts with potent activity within the range.Although such extracts may likely demonstrate cytotoxicity at higher concentrations, the focus in this study was limited to plant extracts that caused substantial growth inhibition in a given cell line within the test concentration of < 100 μg/mL.The assumption was that such activity in the plants crude nature would be indicative of even greater inhibitory effects in the purified state.For initial identification of extracts with activity, the effects were evaluated against human lung NCI-H460 and human cervical HeLa cancer cell lines.

Conclusion
This study has demonstrated the screening process of bioactive extracts with anticancer activity by eliminating the less active ones on the basis of cytotoxicity that takes effective dosage into consideration.Results obtained from extracts screened showed that all extracts exhibited a promising cytotoxic activity against human lung cancer cell line.Among the active extracts and fractions, the fraction with the highest anticancer activity was from the DCM fraction of leaf ethanolic extract, which was found to possess a potent anti-cancer activity with GI50: 30.6 µg/mL.Efficacy and mechanisms of action in various normal and cancer cell models coupled with bioassay-guided purification to identify active anticancer compound(s) from the crude extract will be useful.

Table 1 .
Cytotoxicity dose response of A. calcarata extracts and oils on human lung cancer cell line NCI-H460

Table 2 .
Cytotoxicity dose response of solvent diluted fractions of extracts of A. calcarata on human lung cancer cell line NCI-H460

Table 3 .
Effect of A. calcarata rhizome and leaf ethanolic and water extract, fractions and oils on cervical cancer cell line HeLa Data represented as mean±SE (n=3); %Net growth superscripted by different letters is significantly different at p < 0.05.