Effect of the Water Extracts of Avocado Fruit and Cherimoya Leaf on Four Human Cancer Cell Lines and Vicia Faba Root Tip Cells

The main objective of this study is to determine the effect of the water extract of Persea americana Mill. (avocado fruit) and Annona cherimolia Mill (cherimoya leaf) on living cells. The antiproliferative properties of avocado fruit water extract (AFWE) and cherimoya leaf water extract (CLWE) were determined using four human cancer cell lines: lung (A549), liver (HepG-2), colon (HT-29) and breast (MCF-7). Cancer cells were incubated with 100 μg/ml of AFWE or CLWE for 48 hours, then the cell viability was measured using colorimetric tetrazolium cleavage test (MTT). Both extracts resulted in more than 90% mortality in treated cells. Vicia faba root tip assay was used to determine the effect of AFWE and CLWE on mitotic index (MI), Micronuclei (MN) formation rate and chromosomal abnormalities. Vicia faba roots were soaked in 100, 1250, 2500, 5000, 10000, 20000 μg/ml of AFWE or ALWE for 4 and 24 h. AFWE and CLWE were mitodepressive and resulted in a significant decrease of MI in a dose dependant manner. CLWE treatment resulted in a decrease in prophase cell percentage and an increase in MN & chromosomal abnormalities. On contrary, the prophase cell percentage was linearly increasing with the applied concentration without micronuclei formation in avocado treatment. Our results strongly indicate that avocado and cherimoya extracts were highly cytotoxic and mitodepressive on cancer and plant cells, respectively.

The study of natural bioactive compounds in plants has required the development of bioassay techniques.It is not always possible to test against cancer in animal models due to the system complexity.In vitro methods allow the screening of large numbers for cytotoxicity against many types of cancer cell lines and usually require less test material, time and money (El-Menshawi et al., 2010).
Plant bioassays that measure mitotic cell cycle, micronuclei induction rate and the frequency of chromosomal aberrations are both time and cost effective.These tests are helpful in screening the bioactivity of plant extracts at large scale, particularly in areas with limited funds.They will also eliminate the hazards of using cultured human cells and experimental animals.Plant bioassays are mainly used in most ongoing research to determine the genotoxic effect of chemical compounds rather than testing their pharmaceutical properties.
Root tip meristems of Vicia faba (broad beans) have been used as a pioneer cytogenetic material for the detection of genotoxicity in many studies (Dong & Zhang, 2010;Ma, Cabrera, & Owens, 2005).This bioassay was validated by the International Programme on Chemical Safety (IPCS, WHO) and the United Nations Environment Program (UNEP).As far as the authors are aware, Vicia faba root tip assay had not been used for screening natural plant extracts for their anticancer potential.
Persea americana Mill.(avocado) is an economically important tropical tree belonging to family Lauracea.From phytochemical perspective, extracts of the plant yield functionalized alkanols known as aliphatic acetogenins (Kawagishi et al., 2001).Avocado is used in treating tumor in ethnomedicine and exhibits a chemoprotective effect on human cells (Paul, Kulkarni, & Ganesh, 2011).
Avocado and cherimoya are known to posses stronger cytotoxic effect in tumorous than in normal cells.Some studies showed that plant and animal cells exhibited similar responses towards treatments with bioactive compounds (Konuk, Liman, & CİĞERCİ, 2007).To determine of this is the case, we tested the cytotoxic effect of avocado fruit water extract (AFWE) and cherimoya leaf water extract (CLWE) on four human cancer cells and meristematic plant cells.The bioactivity of the water extracts of avocado and cherimoya had never been tested on human cancer cell lines or plant cells else where.Therefore, this study will be very helpful in highlighting the activities of both extracts and also to compare between the efficiency of the two bioassays in determining their bioactivity.For this aim, the anticancer properties of ALWE and CLWE were determined using four human cancer cell lines: A549 (human lung carcinoma), HePG2 (human liver carcinoma), HT29 (human colon carcinoma) and MCF-7 (human breast carcinoma).The genotoxic effects of the two extracts were determined by measuring the mitotic index (MI), micronuclei (MN) formation rate and chromosomal abnormalities in Vicia faba root tip cells.

Plant Materials
Fresh samples of Persea americana fruit and Annona cherimoli leaves were obtained from El Orman Botanical Garden (Giza, Egypt).The plants were identified by the herbarium officer of El Orman Garden: Mrs Teriz Labib.Voucher specimens were kept in the herbarium, Botany Department, Faculty of Science, Ain Shams University, under the name of Noha-Dina (2009).Leaves were collected at approximately similar age (the third fully expanded leaves from the top of each branch) and fully ripened dry fruits of P. americana were used.Seeds of Vicia faba (cultivar: Windsor white) were kindly provided by the Agriculture Research Centre, Ministry of Agriculture, Giza, Egypt and was used as a reference model in the root tip bioassay.

Preparation of Extracts
Water extracts were prepared by dissolving 2 grams of fresh material in 100 ml of 90ºC distilled water, incubated at the same temperature for 15 min, cooled, filtered and used as a stock (2000000 µg/ml).Water extracts were prepared and used the same day of collection to avoid any change in its activity.

Root Tip Preparations and Treatment
Vicia faba dry seeds were rinsed and then soaked in distilled water for 24 h.Germinating seedlings were kept at 25ºC on moist gauze until their primary roots were 1-2 cm in length (Cotelle, Masfaraud, & Férard, 1999).Root tips were soaked for 4 or 24 h in different concentrations of water extracts (100, 1250, 2500, 5000, 10000, 20000 µg/ml).Distilled water was used as negative control.All treatments were done in triplicates at temperature of 25 ± 2°C.

Cytological Study and Slide Preparation
Root-tips were cut directly into Carnoy's solution [absolute alcohol: glacial acetic acid (3:1)] and incubated at room temperature for 24 h.Roots are then hydrolyzed in 1N analar HCl at 58-60°C for 10 min (Qian, 2004).Root tips-3 mm long-were squashed and stained according to Darlington and La-Cour (1976) Leuco-basic Fuchsin technique.Light green dye (0.3%) was used for background staining of the cytoplasm.Root tips were squashed in 45% acetic acid.Dehydration was done using ascending series of ethyl alcohols; 30%, 50%, 70%, 96%, absolute alcohol; absolute alcohol: xylene (1:1) and xylene, keeping root tips for 5 min in each concentration.Preparations were mounted in Canada balsam and placed at 45°C in the oven (until completely dry).Root tips were examined for micronuclei frequencies and other abnormalities at 4000x magnification using Leica light microscope.The photomicrographs were taken from the prepared slides using digital camera (Sony, 8Mp).

Mitotic Indices (MI) were Calculated Using the Following Formula:
Mitotic index (MI) =

Statistical Analysis
Data shown are the means and standard errors of three or more independent experiments.Statistical comparisons between groups were made by Student's t-test using Microsoft excel program, and a P-value<0.05considered to be statistically significant.

Cytotoxic Effect of Plant Extracts on Cancer Cell Lines
Avocado fruit water extract (AFWE) exhibited strong cytotoxic effect against all cancer cell lines used in this study (Table 1).The extract resulted in lethal percentages of 93.3%, 98.3%, 97.8% and 91.7% in A-549 HepG-2 HT-29 and MCF-7 human cancer cell lines, respectively.The cytotoxic effect of avocado extract was more pronounced as compared to that of cherimoya leaf water extract (CLWE) in HepG-2 and HT-29 cancer cell lines.The cytotoxic concentrations of the two extracts were also tested over a range of dilutions on the four cancer cell lines to determine their LC 50 (Table 2 and Figure 1).Both P. americana and A. cherimolia water extracts showed LC 50 value less than 30 µg/ml in HepG-2 cell lines with LC 50 =13.3& 10 µg/ml and HT-29 cell line with LC 50 = 13.3 & 16 µg/ml, respectively.

The Effect of Plant Extracts on Mitotic Indices
As shown in Table (3), we observed a decrease in the mitotic indices of root tip cells of Vicia faba in a dose-dependant manner in all plant extracts used.Differences in mitotic indices between the treated plants and the untreated control were significant at the highest concentrations of 10000 and 20000 ppm, applied for 4 h, for all treatments applied.In 24 h treatments, the mitotic indices were significantly reduced as compared with control at concentrations of 5000, 10000 and 20000 ppm.

Micron
The water higher con micronucle detected in 24 h (Tabl

Discussion
Many studies, including our own, showed that phytochemicals extracted from Persea americana (avocado) and Annona cherimolia (cherimoya) can selectively induce cell cycle arrest, inhibit growth and enhance apoptosis in some cancer cell lines other than those used here (Ambrosio, Chunhua, Li, Kinghom, & Ding, 2011;Ding, Chin, Kinghorn, & D'Ambrosio, 2007;Garcia-Aguirre et al., 2008).For instance, in vitro studies had shown that the acetone extract of avocado fruit inhibited the growth of prostate cancer cells (Lu et al., 2005) while its chloroform extract inhibited the growth of oral cancer cells (Ding et al., 2009).Fractions from the ethanol extract of cherimoya inhibited the growth of human colon cancer and increased the micronuclei formation in mice erythrocytes (Garcia-Aguirre et al., 2008).However, the effect of avocado fruit water extract (AFWE) and cherimoya leaf water extract (ALWE) have not been reported before either on cancer or plant cells, as far as the authors are aware of.This prompted us to test the properties of AFWE and ALWE in order to mimic the natural way of their consumption by humans.This was carried out using two bio-screeing approaches: 1-cancer cell MTT and 2-Vicia faba root tip -bioassays.
In order to measure their cytotoxic properties, Four cancer cell lines [lung (A549), liver (HepG-2), colon (HT-29) and breast (MCF-7)] were incubated with 100 µg/ml of AFWE or CLWE for 48 h.The Viability of cancer cells was measured using MTT assay.Both extracts resulted in more than 90% growth inhibition in all cancer cell lines as shown in Table 1.This indicates that AFWE and CLWE posses the ability to arrest the growth of cancer cells used in this study.To determine the concentration at which AFWE and ALWE can inhibit 50% of cancer cell growth, we treated all cell lines with series of diluted concentrations and measured cell viability using MTT assay.LC 50 values are shown in Table 2 for both extracts.Avocado and cherimoya water extract exhibited low LC 50 = 13.3 & 10 µg/ml in HepG-2 and LC 50 = 22 & 16 µg/ml in HT-29 cell lines, respectively.Therefore, avocado and cherimoya represent very promising sources for anticancer drugs at least for liver and colon cancers.In the second approach, we used plant system to determine the effect of AFWE and CLWE on mitotic index (MI), micronuclei (MN) formation rate and chromosomal abnormalities.MI, MN and chromosomal abnormalities are well recognized markers to measure the mitodepressive and genotoxic properties of a test substance using plant cells.To date, there are no published data assessing the effect of avocado or cherimoya on Vicia faba root tip cells.Roots of Vicia faba plants were treated with 100, 1250, 2500, 5000, 10000, 20000 µg/ml of AFWE or ALWE for 4 and 24 h.We have noticed a decrease in mitotic index values within root-tips treated with AFWE or ALWE, as compared with the untreated control (Table 3).This decrease was more pronounced in case of AFWE treatments (Table 3).This indicates that both extracts are mitodepressive.Mitodepressive effect had been assumed to result from the inhibition of cells access to mitosis (Badr & Ibrahim, 1987) which is likely attained by preventing DNA biosynthesis or / and microtubule and chromatin organization (Yüzbaşıoğlu, Ãoenal, Sancak, & Kasap, 2003).This will lead into a slower progression of cells from S (DNA synthesis) phase to M (mitosis) phase of the cell cycle (Blakemore, Boes, Cordell, & Manson, 2013).
The percentages of different mitotic phases (Prophase, metaphase, and ana-telophase) were decreasing in a dose dependant manner in AFWE (4 h) and CLWE (4 and 24 h) treated plants c & d).When plants treated with AFWE for 24 h, the prophase cell percentage was progressively increasing in a dose dependant manner (Figure 1b).Thus, it can be assumed that AFWE may have arrested cell division via interfering with DNA biosynthesis rather than affecting other stages in cell cycle.
The induction of micronuclei (MN) had been used in many studies as an indicator of genotoxicity (Cavas & Ergene-Gozukara, 2005).In this work, the genotoxic effect of AFWE and CLWE was determined using the frequency of micronuclei (MN) formation.This will give us an idea about the safety of using these extracts in cancer therapies in future applications.We observed that the decrease in the mitotic index was associated with a significant increase in micronuclei (MN) formation rate in ALWE treated plants at all concentrations used (Table 4).We also observed the formation of two micronuclei per cell (Figure 2 a) and nuclear bud (Figure 2 b) at higher concentrations of CLWE (10000 and 20000 µg/ml).Interestingly, no micronuclei were detected in plants treated with all concentrations of AFWE.
Chromosomal stickiness was the only abnormality observed at a significant level in AFWE treated plants.This gives an indication that the mitodepressive effect of avocado is more potent to an extent that it may had prohibited further cell division.On contrary, the clastogenic effect of cherimoya included chromosomal stickiness, bridges, laggards and disturbed metaphase.In this connection, the presence of micronuclei is commonly associated with structural and numerical chromosomal aberrations induced by clastogenic agents that cause chromosomal breaks and aneugenic agents that disturb microtubule (Bellini et al., 2006;Bellini et al., 2006;Benfenati et al., 2009;Dufour, Kumaravel, Nohynek, Kirkland, & Toutain, 2006).This result suggests that both extracts interfered with the cell cycle.It can also be assumed that CLWE may be affecting chromatin and microtubule organization as indicated by micronuclei formation and chromosomal breaks.The mode of action of AFWE and CLWE as Mitodepressive agents needs further investigation.

Conclusion
This study highlighted the value of avocado fruit and cherimoya leaves to be used as promising sources for anticancer drugs.Both extracts showed potent cytotoxic activity toward cancer cells.However, avocado should be given more attention in this respect due to less chromosomal abnormalities associated with its treatment as compared to cherimoya.
This study can also be used to compare between the efficiency of MTT cancer cell viability and root tip assay in determining the cytotoxic properties of a plant extract toward cancer cells.Our results indicated that both bioassays can lead into the same conclusion.Thus, Vicia faba root tip assay can be used as an initial screening step particularly when large number of extracts is involved.The initial screening can then be followed by extra analysis for the promising extracts to determine the effective dose, selectivity, …… etc using animal models and human cell lines.When it comes to plant bioassay, we found that reduction in mitotic index may indicate the cytotoxic effect of a plant extract on cancer cells.However, MN frequency and chromosomal abnormalities should be considered to validate the bio safety of the extract under investigation.
Figure increas

Figure 4 .
Figure 4. Mean values of different mitotic phases of treated and untreated V. faba root tip cells.Effect of water extract of P. americana (a): for 4 and (b): 24 h.Effect of the water extract of A. cherimolia leaves (c): for 4 and (d): 24 h

Table 2 .
LC 50 values related to MTT assay of the water extract of avocado fruit and cherimoya leaf on human cancer cell lines.Values are in µg/ml Figure 1.LC 50 of the water extract of Persea americana fruit and Annona cherimolia leaves in relation to the four cancer cells lines

Table 3 .
The effect of plant extracts on the mitotic index of Vicia faba L.