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Antiproliferative effect of Viola tricolor on neuroblastoma cells in vitro.

Introduction

Neuroblastoma is the most common extracranial malignant and deadly solid tumor of childhood (Brodeur 2003, Fisher 2012). It originates from the primordial neural crest cells and arises from either the adrenal or anywhere along the sympathetic chain (Dhir 2010). Although most patients have a good prognosis, some infants with MYCN-amplified (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) or MYCN) neuroblastoma show poor survival rates. Therefore the search for new alternative or synergistic antitumor agents has continued (Canete 2009).

Medicinal plants have always been a good source for finding new remedies for human health problems. Many traditional medicinal plants have been tested for their antitumor potential in cell culture or in animal models (Eli 2012, Sakarkar 2011, Tavakkol 2006). They show anticancer effects through inhibiting cancer-activating enzymes, promoting DNA repair, stimulating production of protective enzymes, enhancing body immunity and inducing antioxidant action (Sakarkar 2011).

Viola tricolor, a member of Violaceae plant family, is a common horticultural plant in Iran. It has been reported to have a number of medicinal attributes including anti-inflammatory (Toiu 2007), antimicrobial (WitkowskaBanaszczak 2005), antioxidant (Vukics 2008) and diuretic (Toiu 2009) activity.

Materials and methods

Drugs and chemicals

Mouse neuroblastoma (N2a) cell was obtained from Pasteur Institute (Tehran, Iran). Dimethyl sulfoxide (DMSO), penicillin-streptomycin and 3-(4,5-Dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma (USA). Dulbeccos Modified Eagles medium (DMEM) and fetal bovine serum (FBS) were bought from GIBCO (USA).

Preparation of V. tricolor crude extract

The V. tricolor aerial parts of the flowering plants were collected from Pardis Campus (Mashhad, Iran). The identity of the plant was confirmed and for future reference a voucher specimen (12568) was deposited at the School of Pharmacy herbarium (Mashhad University of Medical Sciences, Iran). The plant materials were dried, powdered and subjected to extraction with 70% ethanol in a Soxhlet apparatus for 48 h. The hydroalcoholic extract was then dried on a water bath and the yield dissolved in DMSO.

Fractionation of hydroalcoholic extract

For preparation of fractions, the dried hydroalcoholic extract (10 g) was suspended in distilled water and transferred to a separator funnel. With solvent/solvent extraction, it was fractionated using ethyl acetate and n-butanol. The ethyl acetate and n-butanol fractions were separated to obtain water fraction (Ghorbani 2012, Sadeghnia 2012). The fractions were dried on a water bath and stock solutions made up in DMSO (ethyl acetate and n-butanol fractions) or saline (water fraction).

Cell culture and treatment

The N2a cells were cultivated in high glucose DMEM supplemented with 10% FBS and penicillin (100 units/mL) and streptomycin (100 [micro]g/mL) at 37[degrees]C in an atmosphere of 5% C[O.sub.2]. Trypsin solution was used to passage cultures whenever they were grown to confluence. The cells at subconfluent stage were harvested from culture flask and after checking the viability with trypan blue exclusion technique they were seeded overnight in 96 well culture plate. To test the possible cytotoxicity of V. tricolor, the culture media was changed to one containing varying concentrations (100-800 [micro]g/mL) of the hydroalcoholic extract and its fractions. The cells were further incubated for 24 h and observed under light inverted microscope for shape, granulation and suspension (anchorage independency).

MTT assay

The effect of V. tricolor on N2a cell proliferation was determined using MTT colorimetric assay as previously described (Hajzadeh 2007, Mousavi 2010). Briefly at the end of treatment the MTT solution was added to each well of culture plate to make a final concentration of 0.5 mg/mL and the reaction mixture incubated for 2 h. The mixture was removed and the resulting formazan dissolved by adding 200 [micro]g/mL L DMSO to each well. The optical density of formazan dye was read at 545 nm (against 620 nm as background). The assay was carried out in triplicate and repeated twice for confirmation. The percentage of viable cells was calculated as the mean [+ or -] SEM with controls set to 100%.

Statistics

All results are presented as mean [+ or -] standard error of the mean (SEM). The values were compared using the one way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. The p-values less than 0.05 were considered to be statistically significant.

Results

Effect of hydroalcoholic extract on surviving cells

As shown in Figure 1 the percentage of surviving cells in all concentrations of the hydroalcoholic extract demonstrated no significant change compared with the control cells. In the presence of 0, 100, 200, 400 and 800 Lg/mL of this extract, the percentage of cell viability was 100 [+ or -] 10, 85 [+ or -] 7.6, 100 [+ or -] 7, 91 [+ or -] 9 and 77 [+ or -] 3.5 respectively.

Effect of water fraction on surviving cells

The presence of water fraction in the culture medium led to a concentration dependent decrease in cell viability (Fig 2). But the effect was not statistically significant even at the highest concentration tested in this study.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The percentage of all surviving cells (quantified by MTT assay) was normalised against untreated control cells (0 [micro]g/ mL). Data are mean [+ or -] SEM of two independent experiments performed in triplicate.

[FIGURE 4 OMITTED]

Cells were treated with increasing concentrations of n-butanol fraction for 24 h. *P < 0.001 versus 0.

The percentage of surviving cells (quantified by MTT assay) was normalised against untreated control cells (0 [micro]g/mL). Data are mean [+ or -] SEM of two independent experiments performed in triplicate.

Compared with untreated cells (100 [+ or -] 10%), the water fraction at 100, 200, 400 and 800 [micro]g/mL decreased the cell percentage viability to 89 [+ or -] 12, 90 [+ or -] 7, 84 [+ or -] 3, 77 [+ or -] 4 respectively.

Effect of ethyl acetate fraction on surviving cells

Figure 3 demonstrates the antiproliferative effect of ethyl acetate fraction of V. tricolor. Following incubation ofN2a neuroblastoma cells with 100, 200, 400 and 800 [micro]g/ mL, approximately 12%, 24%, 38% and 87% inhibition in cell growth was observed respectively compared with untreated cells. The effect of ethyl acetate fraction was concentration dependent and at concentration of 400 [micro]g/ ml (P < 0.05) and 800 ng/ml (P < 0.001) had significant difference compared with that of control cells.

Effect of n-butanol fraction on surviving cells

When the n-butanol fraction was evaluated for in vitro antitumor activity against N2a cells, it was found that after 24 h treatment only high concentrations caused significant cytotoxicity (Fig 4). Exposure of the cells to 800 [micro]g/mL of this fraction showed 76% decrease in cell surviving (P < 0.001 compared with untreated cells). No significant effect was found with 100, 200 and 400 [micro]g/mL of n-butanol fraction.

Discussion

Our data for the first time demonstrated that proliferation of neuronlastoma N2a cells is inhibited by V tricolor. This effect was observed only with ethyl acetate and n-butanol fraction, while none of the hydro-alcoholic extract or water fractions could inhibit the cell growth. Regarding the n-butanol fraction, the best antitumor action was observed at the highest dose (800 [micro]g/mL) used in this study. However the effect of ethyl acetate fraction started at the lower concentration (400 [micro]g/mL). Therefore the main component/s responsible for the antitumor effect of V. tricolor are most likely found in ethyl acetate fraction. While the water fraction contains water soluble plant constituents (e.g. glycosides, quaternary alkaloids, tannins) and n-butanol fraction bears low polar agents (e.g. sterols, alkanes, some terpenoids), the ethyl acetate fraction extracts compounds of intermediate polarity such as flavonoids (Seidel 2006, Dominguez 2010, Tian 2011).

Recent studies have characterised high amounts of saponins, mucilages, cyclotides and flavonoids in V. tricolor (Toiu 2009, Vukics 2008a,b, Dominguez 2010, Svangard 2004). Between the agents, existing evidences support the possible role for flavonoids.

It has been reported that they exert growth inhibitory effects on tumor cells cultured in vitro (Kanadaswami 2005). Deschner et al (1991) indicated that rutin and quercetin, two flavonoid components in V. tricolor (Vukics 2008), inhibit azoxymethanol induced colonic neoplasia.

These reports regarding flavonoids are in agreement with our finding that the ethyl acetate fraction has the best antiproliferative effect between the fractions. On the other hand Tang and co-workers (2010) reported that some cyclotides isolated from V. tricolor have cytotoxic activities against U251, A549, DU145, MDA-MA-231 and BEL-7402 cancer cell lines, showing that other constituents may also be responsible for the antiproliferative effect of this plant.

Conclusion

This study has shown that the ethyl acetate and n-butanol fractions of V. tricolor possess significant antitumor effects against neuroblastoma N2a cells. Isolation and purification of the active compound/s may yield novel anticancer agents.

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Seyed Mohsen Mortazavian (1), Ahmad Ghorbani (2) *

(1) Department of Pharmacology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

(2) Pharmacological Research Centre of Medicinal Plants, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

* Corresponding author: ghorbania@mums.ac.ir, fax +985 118 828567
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Title Annotation:Global dispensary
Author:Mortazavian, Seyed Mohsen; Ghorbani, Ahmad
Publication:Australian Journal of Herbal Medicine
Article Type:Report
Geographic Code:7IRAN
Date:Sep 1, 2012
Words:2080
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