Cytotoxic activity and apoptosis induction of avocado Persea americana Mill. seed extract on MCF-7 cancer cell line

Avocado Persea Americana Mill. is a commercially important crop and studies have shown that the pulp may have beneﬁts to cardiovascular health, dermatological health and possibly anti-cancer activity. Avocado seeds have several medicinal properties such as anti-hyperglycemic, antimicrobial, antioxidant and anti-inﬂammation. This study aim to evaluate the effectofavocadoseedextractonviabilityandapoptosisofbreastcancercelllineMCF-7. Theanticancereffectwasevaluated by cytotoxic test using MTT assay and the effect on apoptosis and cell cycle was examined by ﬂow cytometry method. The cytotoxic test showed that chloroform extract had strong cytotoxic activity against MCF-7 cell lines with IC 50 value of 94.87 µg/mL. Furthermore, the chloroform extract was partitioned with methanol and yield of soluble methanol fraction (FLM) and nonsolublemethanolfraction(FTLM).Thecytotoxicactivityofthemethanolsolublefraction(FLM)andnonsolublemethanol fraction (FTLM) against MCF-7 cell lines was increased with IC 50 of 34.52 and 66.03 µg/mL, respectively. Flow cytometry analysis using annexin-V and propidium iodide staining revealed that methanol soluble fraction could induce apoptosis and modulating the cell cycle arrest in MCF-7 cell. This research indicated that avocado seed has a potency to induce apoptosis and as anti-proliferative to MCF-7 cells lines.


Introduction
Cancer is one of the non-communicable disease which had high incidence and mortality rate worldwide. Ineffective treatments a long with high and expensive medications for cancer theraphy were lead to extensive research on drug discovery and drug development. It was generally accepted that fruit and vegetable consumption will be able to reduce the risk of human cancer (La Vecchia et al. 2001). The protective effect of fruit and vegetables is related to an amount of phytochemicals components. Avocado is one of commercial fruit that produce and consume worldwide. Central and South American countries dominate global avocado production. Mexico is the world's leading avocado producer while Indonesia noted as the second highest producer (USAID 2014).
Avocado Persea americana Mill. belong to Lauraceae family is an evergreen tree which native to Central America and presently have been cultivated worldwide. Avocado is grow in a wide range of climate from low land to mountainous region. Avocado is cultivated as delicious and nutritious fruits rather than as medicine. The fruit of avocado contains high percentage of lipids that reach up to 20% of dry weight (Takenaga et al. 2008). A part of nutritional and economical value of its fruits, several folk medicinal properties has been reported from the different organs of P. americana. Avocado is used in treating tumor in ethnomedicine and exhibits a chemoprotective effect on human cells (Paul et al. 2011).
One-half an avocado is a nutrient and phytochemical dense food consisting of dietary fiber, vitamins, minerals, and unsaturated fatty acid that have many health benefit (Dreher and Davenport 2013). Avocado seed is underutilized and represents a large portion of the fruit (Duarte et al. 2016). The seed amount more or less 16% of total avocado weight, and it is still an under-utilized resource (Ramos-Jerz et al. 2013). Phytochemical studies on avocado seeds have identified various classes of natural compounds such as phytosterols, triterpenes, fatty acids, furanoic acids, abscisic acid, proanthocyanidins, and polyphenols (Ding et al. 2007;Leite et al. 2009). Wang et al. (2010) have reported the presence of catechin, epicatechin, and A-and B-type procyanidin dimers and trimers, tetramers, pentamers, and hexamers in the seed of avocado.
Ethnopharmacological studies of the Aztec and Maya cultures have reported the use of decoctions of avocado seeds for the treatment of mycotic and parasitic infections.
Seeds have also been reported for use against diabetes, inflammation, and gastrointestinal irregularity (Dabas et al. 2013). The powdered form of avocado seed has been used for skin eruptions and to cure dandruff (Morton and Dowling 1987). Avocado seeds have more antioxidant activity and polyphenol content than the pulp (Nagaraj et al. 2010). Recent studies have demonstrated the anti-cancer, anti-diabetic, anti-inflammatory, blood pressure reducing, anti-microbial, insecticidal and dermatological activities of seed preparations. The anti-carcinogenic effects of avocado pulp have been investigated. Inhibition of PC3 and LNCaP prostate cancer cell lines was observed after treatment with an acetone extract of the pulp of Hass variety (Ding et al. 2009). In this study, it will observe the effect of soluble (MF) and non-soluble (NMF) methanolic fraction of avocado seed extract on the viability and apoptosis of breast cancer cell line MCF-7.

Plant source
Fresh seeds of avocado were obtained from the Collection Garden of Medicinal Plant and Traditional Medicine Research and Development Centre, at the altitude of 1.200 m above sea level, with andosol type of soil. The fruits were harvested on July 2015, and after ripening in air temperature for six days than it was peel off to collect the seeds. The seeds were sliced into small and thin size, then drying in oven with 50°C of temperature for 3 x 24 h.

Extraction and fractionation
As 1000 g of dried powdered of seed was pulverized, then macerated in chloroform for three days and then filtered. The solvent was removed using a rotary evaporator at 50°C for 6-8 h. In our previous paper, the chloroform extract (EKBP) showed highest activity on MTT assay, therefore CE was chosen for the study (Widiyastuti et al. 2014). The chloroform extract was further partition with methanol and yielded soluble (FLM) and insoluble methanolic fraction (FTLM). All the extracts were examined for the chemicals profiles by spot test and TLC methods (Harborne 1987). FLM and FTLM were evaluated for its cytotoxic activity using MTT assay. The fraction which has more cytotoxic activity was used in this study to evaluate the capability of the extract to induce cell cycle arrest and apoptosis in MCF-7 cancer cell lines.

MTT assay of soluble (FLM) and insoluble methanolic (FTLM) fraction
The cytotoxic tests were carried out using the MTT assay method. MCF-7 were cultured maintained in Modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 µg/mL penicillin and 100 µg/mL streptomycin) and cultured in CO 2 incubator containing 5% CO 2 at 37°C. The cells were seeded at a density of 8 x 10 5 cells in 96-well plates with MEM medium and incubated for 24 h. The cells were treated with extracts of various concentration such as 5, 20, 30, 60, 120, and 200 µg/mL in 0.1% DMSO for 48 h exposure time. After 48 h, 100 µL of a 1 mg/mL of solution of MTT in MEM was added to each well. The culture plates were incubated for 4 h at CO 2 incubator containing 5% CO 2 at 37°C. MTT was removed carefully and then stop solution (HCL in isopropanol) was added to each well and the plate was vigorously shaken to ensure that the blue formazan was completely dissolved. The absorbance was measured at 595 nm in automated plate reader (Elisa Reader Biorad) and percentage of growth inhibition was calculated using Equation 1.
(1) IC 50 was defined as the concentration of the plant extracts killing 50% of the cells. IC 50 was determined for MCF-7 cell lines of both extracts.

Cell cycle evaluation
A number of 5 x 10 5 cells MCF-7 in 1 mL of culture medium EMEM grown in a six-well plate, and adapted for 24-48 h in a 5% CO 2 incubator temperature of 37°C. Once the cells was confluent, than it were treated by ½IC 50 test sample concentration and IC 50 (30 µg/mL). One of the well filled with the culture medium as a control. Observation and documentation of the cells under inverted microscope is done after 24-48 h of incubation. Each treatment was prepared a 15 mL conical. Cell culture media was transferred into conical, washed with 1x PBS and then transferred to the same conical. Cells were harvested with 150 mL of 0.25% trypsin-EDTA which incubated for 3 min. Cells were resuspensioned with 1 mL of culture medium. Remaining cells were taken with PBS and then transferred to a conical tube and centrifuged at 500 rpm for 4 min. The supernatant was discarded; the cell pellet was washed with 500 mL of cold PBS. Further, conical tube was centrifuged at 500 rpm for 4 min. The supernatant was discarded; the cells were fixed with a drop of 500 µL of 70% ethanol into conical tube than shaken slowly and stood for 30 min at room temperature. Conical tube was centrifuged around 2000 rpm for 2 min, than the alcohol removed and cells were washed with 500 µL of PBS and then centrifuged around 2000 rpm for 2 min. PI reagent (20 µL propidium iodide (Sigma) 1 mg/µL + 0.5 µL tritonx (Sigma) 100 + 1 µL RNase (Roche) 10 mg/µL in 500 µL of PBS for each sample) was added to the cell pellet, than resuspensioned and stored in a dark room for 20 min. The cell suspension was transferred into the new tube/micro tube then read and analyzed using BD flow cytometer Accuri C6.

Flow cytometry analysis of apoptosis
Cellular DNA content was determined by flow cytometry analysis of PI-labeled cells. A number of MCF-7 cells of 5 x 10 5 was seeded in 1 mL of culture medium EMEM grown in a 6-well plate, and adapted for 24-48 h in a 5% CO 2 incubator with temperature of 37°C. Once confluent, the cells were treated with ½IC 50 and IC 50 of concentration of avocado seed extract. One of the well was only filled by culture medium as a control. Observation and documentation of the cells under inverted microscope were done after 24-48 h of incubation. Each treatment was prepared a fruit conical 15 mL. Cell culture media is transferred in conical, washed with 1x PBS and then transferred to the same conical. Cells were harvested with 150 µL of 0.25% trypsin-EDTA and incubated for 3 min. Resuspension of cells with 1 µL culture medium. Remaining cells were taken with PBS and then transferred to conical and centrifuged 500 rpm for 4 min. The supernatant was discarded, the cell pellet was added with apoptosis reagent (FITC annexin 3 mL+3 mL of propidium iodide in 100 mL of buffer annexin), left in a dark room at room temperature for 10 min. Cells suspension was transferred to a tube then read and analyzed using BD flow cytometer Accuri C6.

Data analysis
Data analysis to calculate the IC 50 value from single cytotoxicity assay, we plotted linear regression of concentration and percentage of cells viability using Excel MS Office 2013. Combination treatment was evaluated by calculating the Combination Index (CI) value with Compusyn software. The data obtained from flow cytometer was analyzed using BD Accuri C6 software.

Chemical profiles of soluble and non-soluble methanolic fraction of avocado seed extract
Chemical analysis was conducted to determine the different compound contains in the extracts. Qualitative determination of chemical compounds of FLM, FTLM and EKBP extracts showed the different profiles of chromatogram Figure 1.
In the soluble methanolic fraction appeared five spots having Rf value between 0.4 to 0.9, and in the insoluble methanolic fraction appearing three spots on visualization with UV both 254 and 366 nm as well as with reagents.  In this study, the thin-layer chromatographic profile (Figure 1) of the non-soluble fraction indicates the presence of non-polar compound with the Rf value more than 0.5. The insoluble methanolic fraction of avocado seeds showed only less spot on TLC which indicates that almost all of the compounds have been dissolved in methanol.

Inhibitory effect of methanolic fraction of avocado seed extraction MCF-7 cells growth
The chloroform extract was partition with methanol and yielded soluble (FLM) and insoluble methanolic fraction (FTLM). The methanolic soluble fraction (FLM) and FTLM were evaluated for its cytotoxic activity using MTT assay. Cytotoxic test showed IC 50 of FLM and FTL Mon MCF-7 cells growth resulted the decreasing value of IC 50 compare to chloroform extract of avocado seed (EKBP) ( Table 1). Profiles obtained from cell viability by MTT test showed that the FLM and FTLM of avocado seed in general may decrease the viability of MCF-7 cells compared to chloroform extract. The effect of all avocado seed extracts were showed by decreasing of cell viability with dose dependent manner. Cells treated with FLM, FTLM and EKBP for 48 h resulted in growth inhibition and cell death in a dose dependent manner (Figure 2).
Cytotoxic effect is the effect depends on the concentration level of materials where the greater concentration of materials so the greater of the cytotoxic effect levels (Wahyuningsih et al. 2015). From the previous evaluation, avocado seed chloroform extract (EKBP) shown the highest inhibitory activity (Widiyastuti et al. 2014). Active compounds contained in chloroform extract of avocado seed are estimated to have the potential cytotoxic activity of its value IC 50 lower than 100 µg/mL (Table 1).
Furthermore, the effect of FLM and FTLM on cell viability also observed by the morphological changes of MCF-7 cell line. FML causes more cell deaths than FTLM which characterized by morphological changes in the form of cell shrinkage and cell extension. At a concentration of 30 µg/mL, FLM could kill more than 50% of MCF-7 cells (Figure 3).

Effect FLM and FTLM on cell cycle of MCF-7 cancer cell lines
In this study, MCF-7 breast cancer cell lines was used to evaluate the capability of avocado seed methanolic fraction of chloroform extract to induce apoptosis and effect on cell cycle. Observation of cell cycle profile was performed on the soluble and insoluble methanolic avocados seed extract (Figure 3). Treatment of FLM and FTLM extracts did not affect the overall cell cycle profile, but induced an increase in the number of cells in subG1 phase ( Figure 4). Percentage of cell counts in each cycle phase can be seen in Table 2. Both extract are not affected the cell cycle of MCF-7 significantly. The contrast was the FLM extract able to increase the number of cells in SubG1/G0 phase from 5.1% to 21.9% after 48 h of incubation which reflects the occurrence of apoptotic event.

FLM and FTLM of avocado seed induced apoptosis in MCF-7 cell lines
To understand cell death mechanism in subG1 population, whether it was mediated through apoptosis, it was stained treated cells with propidium iodide-annexin and subjected to flow cytometry analysis. The results showed the apoptotic population was significantly increased in MCF-7 cells treated by both avocado seed extract of FLM and FTLM as well ( Figure 5).

Discussion
Avocado are fruits that are usually consumed in our diet and also have compounds that are essential for health, including terpenoid and phenolic substances. Avocado seed is discarded and considered as a waste of product. From the previous study catechin and epicatechin were isolated from a methanolic extract of avocado seed showed antioxidant activity in an AMVN-induced methyl linoleate peroxidation assay (Matsusaka et al. 2003). Avocado seed also rich in polyphenols with antioxidant and antimicrobial power (Rodríguez-Carpena et al. 2011). The chloroform and methanolic soluble fraction of avocado seed have a different compounds indicated by a number and Rf value of the spots. Research showed that qualitative determination of chemical group compound of avocado seed chloroform extract contained alkaloid, saponin, and terpenoid, while the methanolic extract contained alkaloid, saponin, tanin, terpenoid, and flavonoid (Widiyastuti et al. 2014). The differences polarity of the various solvents are perhaps responsible for the differences in the solubility of plants active compound, therefore affect in degree of activity. Alkaloid, flavonoid and terpenoid are the chemical compound groups which possess cytotoxic activity, inhibited cell growth and induced apoptosis in various lines of cancer (Taraphdar et al. 2001). The solvent of extraction has been shown to be an important factor in the extraction of bioactive compounds of avocado seed that lead to different activities. Isolated compound of avocado seed ethanolic extract that identified as triterpenoid was able to inhibit the viability of MCF-7 cancer cell line with IC 50 value of 62.43 µg/mL (Abubakar et al. 2017).
The partition of avocado seed chloroform extract with methanol yielded soluble (Figure 1). The methanolic soluble fraction has contained more polar compound compare to chloroform extract, indicate by the lower of Rf value of the spots. That means the methanolic soluble fraction has more cytotoxic activity since the existence of the polar compounds. According to Elmore et al. (2002), the highest concentration of a test agent in cytotoxicity evaluation should be less than 1.000 μg/mL or 1.000 μM. If none of the concentrations of test agents exhibited cytotoxic effect in excess of 50% of cell populations, the test agent is considered non-toxic against the tested cell line. In the present study, FLM and FTLM extracts at concentrations of 100 μg/mL could reduce the viability of MCF-7 cells to less than 50% (Figure 3). Thus, both extracts were considered toxic against MCF-7 cells. From the results obtained it is known that the compounds responsible for the cytotoxic activity of avocado seeds are more polar.
The measurement of the viability of cancer cell line is one of the parameter to evaluate the anticancer potency of avocado seed extract. Moreover the effect of the extract on cell growth and the induction of apoptosis are also important parameters to evaluate the respective anticancer properties of avocado seed extracts. Therefore, cell cycle analysis was conducted to determine the influence of avocado seed extract on the cell cycle behavior and apoptosis initiation by flow cytometry method. Based on cell cycle profile with flow cytometry, treatment of extract induces apoptosis (sub phase G1 and G2/M) without modulating the cell cycle. FLM was induce sub-G1 phase arrest (21.9%) which reflecting of apoptotic cell death population as compared to FTLM and control cell which only 5.1% and 3.7% respectively (Table 2). Ding et al. (2009) suggest that the chemical compounds present in FLM extract can inhibit the growth of oral epithelial of cancer cell line with the target of protein regulation in the cell cycle. Apoptosis or cell death progam, is a cellular process that occurs in normal or pathological of physiological conditions. Disregulated form of molecular sig-  naling of apoptosis resulting in a proliferation imbalance and cell death which plays an important role incarcinogenesis events. Therefore, induction of apoptosis is one of the mechanisms which contribute importantly in cancer therapy (Wong 2011). Therefore, the molecular mechanism of avocado seed extract in inducing apoptosis of MCF-7 cancer cells needs to be further studied. Cell apoptosis was also increased by FLM dosedependently ( Figure 5). A previous study revealed that Persea americana Mill var. Hass (Lauraceae) was inhibited viability and induce apoptosis in prostate and oral epithelial cancer cell lines (D'Ambrosio et al. 2011;Ding et al. 2009).

Conclusions
The methanol soluble fraction of avocado seed extract has the highest cytotoxic activity on MCF-7 cell lines because it has the lowest IC 50 value of 34.52 mg/mL.The methanol soluble fraction (FLM) and the insoluble methanolic fraction(FTLM) did not significantly affect the cell cycle but FLM extract was increase the accumulated of cell in the subG1 and G2/M phase that indicate the occurance of apoptotic event.