Therapeutic Index of Methanolic Extracts of Three Malaysian Phyllanthus Species on MCF-7 and MCF-10 A Cell Lines

Introduction: Phyllanthus species such as P. urinaria, P. niruri and P. debilis are common herbs found in Malaysia that are traditionally used for treatment of chronic diseases such as liver diseases, kidney stones and cancer. Phyllanthus species were shown in vitro to have many biological functions such as anti-cancer, anti-inflammatory, hepatoprotective and anti-diabetic. Aims: The goal of this in vitro study was to assess the cytotoxic effect of the methanolic extract of P. urinaria, P. niruri and P. debilis on MCF-10A and MCF-7 cells (i.e., normal and cancerous breast cell lines) and to determine the therapeutic index of each Phyllanthus species. Materials and Methods: We determined the therapeutic index for each Phyllanthus sp. and its selective toxicity towards these cells. The toxicity of sample toward the cells are measured by trypan blue cell counting method. Results: Our results showed that P. debilis had the lowest IC50 concentration in MCF-7 cells and the highest IC50 concentration in MCF-10A cells and its therapeutic index was higher than that found in P. niruri and P. urinaria. The high therapeutic index of P. debilis suggests that this species has greater selective cytotoxicity in MCF-7 cancer cells than in MCF-10A normal cells. Thus, the methanolic extract of P. debilis should be further characterised and developed for future use as an anti-cancer agent.


INTRODUCTION
Phyllanthus is a large genus of trees, shrubs and herbs of the family Euphorbiaceae.The genus contains more than 600 species and is commonly found in tropical regions worldwide.¹3][4] The three species most commonly used to treat certain diseases in Malaysia are P. niruri, P. debilis and P. urinaria.6][7][8][9] The aim of this study was to evaluate the selective toxicity of the methanolic extract of P. urinaria, P. debilis and P. niruri in normal (MCF-10A) and breast cancer cells (MCF-7).The trypan blue assay was performed to investigate the cytotoxicity effects of the methanolic extracts of P. urinaria, P. niruri and P. debilis on MCF-7 and MCF-10A cells.Cells were treated for 24 hours with medium containing methanol extracts at different concentrations and the IC 50 and EC 50 were determined for each extract.The therapeutic index was calculated to determine whether the cytotoxic effects of each methanol extract were selective for cancer cells (MCF-7) in comparison to non-cancer cells (MCF-10A).In this study, the therapeutic index is defined as the ratio of the half inhibitory concentra-tion of the extract at which an extract becomes toxic in normal cells and the half effective concentration of the extract at which the drug is effective in cancer cells. 10e determined the therapeutic index of these three Phyllanthus species to identify the species with the highest therapeutic index for future development of a chemotherapeutic agent.

Samples
P. urinaria, P. niruri and P. debilis were harvested from the local collection at Tasek Gelugor, Penang, Malaysia.These species were identified by a botanist from the School of Biological Sciences, Universiti Sains Malaysia (USM).Voucher specimens (Phyllanthus debilis: 11623, Phyllanthus niruri: 11624 and Phyllanthus urinaria: 11625) were deposited at the USM herbarium.

Plant extraction
The whole plant sample were dried and crushed into a powder form.Five grams of sample was then extracted with 100 mL of methanol in an ultrasonic bath for 20 min.After filtering the filtrate, the process was repeated twice with the remaining residual Pharmacognosy Journal, Vol 10, Issue 6 (Suppl), Nov-Dec, 2018 S31 extract.The methanolic extract was then dried using a Buchi Rotary Evaporator RII (Switzerland) and then freeze dried using FDU-1200 Eyela device (USA).The dried extracts were stored at -2 to -8°C for future use.

Cell Culture
Human breast carcinoma (MCF-7) and human breast epithelial (MCF-10A) cell lines were used in this study.MCF-7 cells were grown in RPMI-1640 medium (Gibco) and MCF-10A cells were grown in Dulbecco Modified Eagle Medium.The medium for MCF-7 cells was supplemented with 10% fetal bovine serum (Gibco) and 1% penicillinstreptomycin (Gibco) and incubated in a humidified atmosphere with 5% carbon dioxide at 37°C.The medium for MCF-10A cells was supplemented with 1% hydrocortisone (Sigma-Aldrich), 1% penicillin-streptomycin (Gibco), 10% horse serum (Gibco), 0.25% human recombinant insulin (Gibco) and 0.002% recombinant human epidermal growth factor (Gibco) and incubated in a humidified atmosphere with 5% carbon dioxide at 37°C.There were two technical replicates for each experiment and each experiment was repeated three times.

Trypan blue cell counting
Cells were seeded at cell density of 0.05 x 10 6 cells/well in 24-well plates and incubated in 5% carbon dioxide at 37°C for 36-48 hours to allow cell attachment and to reach 70% confluency.Once the cells reached 70% confluence, the cells were treated with five different concentrations of the extracts.Both methanolic extract and aqueous extract sample were dissolved in growth medium.The concentration for methanolic extracts ranged from 31.25 to 500 µg/mL, while for aqueous extract, the concentration ranged from 62.5 to 1000 µg/mL.The control wells were prepared with cells without treatment.The half inhibitory concentration in normal cell (IC 50 ) and half effective concentration in cancer cells (EC 50 ) of the extract was determined for MCF-7 and MCF-10A by determined the concentration of extract at which 50% of the target is inhibited.Cells were incubated at 37°C in 5% carbon dioxide for another 24 hours.The cells then were washed using phosphate buffered saline (PBS) and incubated with trypsin for 5 min to detach the cells from the well.Medium was added to the well to deactivate the trypsin and the mixture was thoroughly mixed.The cell suspension was then mixed with 0.4% trypan blue solution in the ratio of 1:1 and 10 µL of this suspension were directly counted using a haemocytometer.The average cell count of four fields was used to represent the number of cells per mL of cell solution, it and was used to determine the total number of cells from each well.The number of cells was counted in each of the four quadrants using the following formula: Number of cells = ((A + B + C + D) / 4) × 10 4 × dilution factor × sample dilutions.The viability of the cells was determined by comparing the number of viable cells in the treatment with that in the untreated group as follows: Percentage of viable cells (%) = (Number of viable cells from treatment / Number of viable cells (untreated)) × 100.

Calculation of therapeutic index
Therapeutic index was calculated by dividing the IC 50 (inhibition concentration of 50% in normal MCF-10A cells) to EC 50 (inhibition concentration of 50% in MCF-7 cancer cells).IC 50 and EC 50 were extrapolated from the graphs constructed based on cytotoxicity assay using Microsoft Excel 2016 for Windows.

RESULTS AND DISCUSSION
Therapeutic index provides a simple index for assessing the safety and efficacy of target drugs.Extracts with high therapeutic index are effective at killing cancer cell at a lower concentration (i.e., the lower EC 50 concentration used in cancer cells compared to the higher IC 50 concentration in normal cells) than extracts with low therapeutic index. 11Table 1 shows the IC 50 , EC 50 and therapeutic index of each Phyllanthus species.In the MCF-7 cancer cells, the methanolic extract of P. debilis had the lowest EC 50 concentration, followed by P. niruri and P. urinaria.In normal MCF-10A cells, P. debilis had the highest IC 50 concentration, followed by P. niruri and P. urinaria.Thus, P. debilis had the highest therapeutic index, followed by P. niruri and P. urinaria (Table 1).Higher therapeutic index indicates more selectivity towards cancer cells than normal cells.These results suggest that P. debilis has stronger selective cytotoxicity towards MCF-7 cancer cells but lower toxicity to normal MCF-10A cells compared to the other Phyllanthus species tested.In contrast, P. urinaria showed indistinct cytotoxic activity in both MCF-10A and MCF-7 cells.It also had the lowest therapeutic index, suggesting that the methanol extract of P. urinaria was the most toxic to MCF-10A cells.However, Lee et al., (2011) reported that the methanolic extract of P. urinaria showed better selective toxicity in MCF-7 cells than in normal breast epithelial cells (184B5), with EC 50 in MCF-7 cells of 48 µg/ mL compared to IC 50 at > 500 µg/mL in normal 184B5 cells. 5This discrepancy between studies might be because different normal breast cell lines were used (MCF-10A and 184B5) and the P. urinaria methanolic extract might affect them differently.The biochemical and biological responses of those cells (MCF-10A and 184B5) are highly variable and the effect of the extract might depend on genetic alterations present in those cells. 12Bio-guided assay development and analysis of the compounds present in the methanolic extract are needed to identify the possible compounds responsible for the cytotoxic effect seen in MCF-10A cells.

CONCLUSION
Methanolic extracts of P. urinaria, P. niruri and P. debilis shows different toxicity towards normal breast cell lines and breast cancer cell lines.This study shows that P. debilis had highest therapeutic index, followed by P. niruri and P. urinaria.This means that this species is more effective in killing cancer cell at a lower concentration.Further studies are warranted for better understanding of their biological mechanisms.