In vitro evaluation of cytotoxic activity of flower, leaf, stem and root extracts of five Artemisia species.

The present study was carried out to investigate cytotoxic activity of flower, leaf, stem and root extracts of five Artemisia species against breast cancer cell line (MCF7) and human embryonic kidney normal cell line (HEK293). The studied Artemisia species were A. absinthium, A. vulgaris, A. incana, A. fragrans and A. spicigera. The cytotoxic activity was measured by MTT assay at different concentrations (62.5, 125, 250, 500 μg/ml). Among these five species, methanol extracts of flower, leaf, stem and root of A. absinthium and A. vulgaris exhibited considerable cytotoxic activity. The flower extracts of these two species were found to have higher cytotoxic effect on MCF7 cell with an IC50 value of 221.5 and >500 μg/ml, respectively. Leaf methanol extract of A. incana also showed cytotoxic activity. Cytotoxic activity of different extracts of A. absinthium, A. vulgaris and A. incana against MCF7 was 10%-40% more than HEK293 cells. Not only the extracts of A. spicigera and A. fragrans did not show any cytotoxic effect against both cell lines, but also increased the number of cells. This study revealed that A. absinthium and A. vulgaris may have a great potential to explore new anticancer drugs.

INTRODUCTION

Artemisia is a shrub or small herb that grows in dry and semi-dry regions. This genus chiefly is found in northern hemisphere and lowers in southern hemisphere. The genus Artemisia belongs to the Anthemideae tribe of Asteraceae family. There are about 500 species of herbs and shrubs in this genus (1). Artemisia species are mainly found in Asia, Europe and North America (2).

The greatest number of these species was attained in Asia (3). Thirty five species of this genus are found in Iran (4). The Artemisia species have been used in Iranian traditional medicine as anti-infectious, anti-bacterial, gastric tonic, digestive and stomachic (5). Recently, monoterpenes, sesquiterpenes, sesquiterpene lactones, flavonoids, coumarins, sterols, polyacetylenes have been isolated from Artemisia species (678). Previous studies on some Artemisia species have shown that most species possess medicinal properties such as anti-bacterial and anti-cancer effects (9101112). Many in vitro and in vivo studies have been published on the anticancer activity of different species of Artemisia (131415).

Some studies reported that fresh leaves of A. absinthium and flowers of A. vulgaris have strong cytotoxic effect against breast cancer cell line (MCF7) (1617). But there is no scientific study available about anti-cancer activities of flower, leaf, stem and root of A. absinthium and A. vulgaris. The present study is focused to evaluate the cytotoxic activity of methanol extract prepared from various organs of Artemisia species on MCF7 cell line and human embryonic kidney normal cell line (HEK293).

MATERIALS AND METHODS

Plant material

Five species of Artemisia (A. absinthium, A. vulgaris, A. fragrans, A. spicigera and A. incana) were collected from two different regions in Urmia city, West Azerbaijan Province, Iran, in October 2011. These species were recognized after a series of taxonomic revisions. The Voucher specimens were deposited at the Herbarium of National Botanical Garden of Iran.

Preparation and analysis of extract

The samples were separated into flower, leaf, stem and root parts. The plant material was carefully dried in shadow and powdered. The dried plant samples (30 g) were separately placed in a stopped conical flask and macerated with 250 ml methanol (98% v/v. Merck, Germany) at room temperature (25–28°C) for 72 h with occasional stirring. The solvent was filtered and evaporated in a vacuum rotary evaporator (Stroglass, Italy) at 45°C. The residue was placed in freeze drier (Zirbus, Germany) to dry. The crude extract was stored in a well-closed container, protected from light and kept in a refrigerator at 4°C. 40 mg of the sample extract were dissolved in 1 ml 100% (v/v) of dimethyl sulfoxide (DMSO). Each experiment was performed in triplicate.

Cell lines and culture medium

Cell lines were purchased from the Cell Bank of Pasteur Institute in Tehran (Iran). MCF7 and HEK293 cells were cultured in Roswell Park Memorial Institute (RPMI-1640) and Dulbecco's Modified Eagle Medium (DMEM) respectively and supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM/L glutamine and 1 mM sodium pyruvate. All of the reagents were purchased from Gibco (Scotland).

Cytotoxicity assay

The cellular toxicity of the methanol extracts of five species of Artemisia on cultured cells were measured using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (18).

The cells were grown in 96-well plates at a density of 5 × 104 cells per well. After 2 h, cells were treated with different concentrations of samples (62.5, 125, 250, 500 μg/ml) and incubated for 48 h. Later, 25 μl of the MTT solution (5 mg/ml) was added to each well, and the plate was reincubated for 2 h. Finally, the medium was removed and 100 μl of DMSO was added to solve formazan crystals.

The amount of formazan crystal was determined by measuring the absorbance at 570 nm by using a micro plate spectrophotometer (Awareness Technology Inc., stat fax 2100).

The survival curves of each cell line were established based on extract concentration after the specified period.

Statistical analysis

Each experiment was performed in triplicate and repeated two times. The experiments were performed using completely randomized design (CRD) and results were analyzed using one way ANOVA. Statistical analyses were performed by using Software SAS, Version 6.12. Probability P<0.05 were considered significant.

RESULTS

The different parts of five species of Artemisia were tested for cytotoxicity against MCF7 and HEK293. All extracts from species were tested under comparable conditions at different concentrations (62.5, 125, 250, 500 μg/ml).

The plant extracts were included flower, leaf, stem and root. Among these five species, methanol extracts of different organs of A. absinthium and A. vulgaris strongly exhibited cytotoxic activity against MCF7 (Figs. 1, 2). The flower extracts of A. absinthium and A. vulgaris were shown higher cytotoxic effect against MCF7 cell with an IC50 value of 221 and >500 μg/ml, respectively.

Fig. 1

Cytotoxic effect of flower, leaf, stem and root extracts of A. absinthium against A: MCF7 and B: HEK293 cells. Cell viability was assessed using MTT assay. Data are shown as Mean ± SD, n=3.

Fig. 2

Cytotoxic effect of flower, leaf, stem and root extracts of A. vulgaris against A: MCF7 and B: HEK293 cells. Cell viability was assessed using MTT assay. Data are shown as Mean ± SD, n=3.

The cytotoxicity was followed by leaf, stem and root extracts with IC50 343, 430 for A. absinthium and IC50>500, μg/ml, for A. vulgaris respectively.

Based on these results, different organs of A. absinthium had stronger activity than A. vulgaris. Methanol leaf extract of A. incana showed cytotoxic activity at higher concentration (500 μg/ml) which decreased cell viability up to 32% and the other organs did not have any cytotoxic effect (Fig. 3). In spite of above results, different parts of A.spicigera and A. fragrans significantly increased the number of MCF7 and HEK293 cell lines (Figs. 4, 5). So that methanol extract of root, leaf, flower and stem in A. spicigera increased MCF7 proliferation up to 93%, 86%, 79% and 60% respectively and similarly A. fragrans increased the number of cancer cells up to 54%, 50%, 31% and 11% at concentration of 500 μg/ml.

Fig. 3

Cytotoxic effect of flower, leaf, stem and root extracts of A. incana against A: MCF7 and B: HEK293 cells. Cell viability was assessed using MTT assay. Data are shown as Mean ± SD, n=3.

Fig. 4

Cytotoxic effect of flower, leaf, stem and root extracts of A. spicigera against A: MCF7 and B: HEK293 cells. Cell viability was assessed using MTT assay. Data are shown as Mean ± SD, n=3.

Fig. 5

Cytotoxic effect of flower, leaf, stem and root extracts of A. fragrans against A: MCF7 and B: HEK293 cells. Cell viability was assessed using MTT assay. Data are shown as Mean ± SD, n=3.

DISCUSSION

The results showed that methanol extract of different parts of A. absinthium and A. vulgaris has strong cytotoxic activities against MCF7. Aqueous, methanol and acetone extracts of A. absinthium have been reported to possess excellent anticancer activity against MCF7 by IC50 values of 244.9, 14.31 and 6.11 μg/ml respectively (16). Nawab and coworkers, observed that A. vulgaris has significant dose-dependent inhibition of the proliferation and viability of the cancer cells such as colon carcinoma (RKO), prostate cancer (PC3) and human breast cancer (T47D) (17). The other species like A. princeps have also been reported to inhibit the growth of breast cancerous cells generally in a concentration dependent way (12).

Phytochemicals screening of the methanol extract from aerial parts of A. vulgaris and A. absinthium revealed the presence of tannins and flavonoids (such as eupafolin, diosmetin, rhamnetin, apigenin and their glucosides, luteolin, quercetin, rutin and vitexin) which are characterized for their anticancer properties (1719). Nibret and coworkers reported that the aerial parts of A. absinthium have cytotoxic activity against human promyelocytic leukemia cells (HL60) (20).

In the present study, flower extract of A. vulgaris and A. absinthium have the most cytotoxicity against MCF7 cells compared to those of leaf, stem and root extracts. It may be due to accumulation of artemisinin in flower extract of these two species in comparison with stem and roots. It has been reported that flower and leaf extracts of some Artemisia species are rich sources of artemisinin (21).

It has also been revealed a good correlation between the artimisinin accumulation in different parts of Artemisia species and its anticancer activity. artemisinin is a sesquiterpen lactones and demonstrated anti-cancer activities when tested in vitro and in vivo (22). Results of present research showed that cytotoxic activity of different parts of A. absinthium is more than that of A. vulgaris. The higher cytotoxicity of A. absinthium could be due to the high content of artemisinin in flowers, leaves, stems and roots which have been reported before (21).

In this work, the results of MTT assay showed that A. spicigera and A. fragrans have an increasing effect on number of MCF7 and HEK293 cell lines.

This increasing effect is due to occurrence of some compounds in these two species that can stimulates cell proliferation. Previous studies have reported that some ingredients in natural products can significantly increase proli-feration of cancer and normal cell lines. The low concentration of essential oils isolated from Artemisia princeps has been shown to possess an increasing effect on proliferation of endothelial cells (23). Le Bail and coworkers, reported that flavonoids at low concentrations can significantly enhance the proliferation of human breast cancer cells MCF7 (24). These results indicated that the essential oils and flavonoids contained some low molecular-weight components stimulates the proliferation of vascular endothelial cells in vitro (25).

CONCLUSION

In conclusion, these findings demonstrated flower extracts of A. absinthium and A. vulgaris and leaf methanol extract of A. incana can be used as cytotoxic agents, however, further investigations have to be performed.