Cytotoxic Potential of Petroleum ether, Ethyl Acetate, Chloroform, and Ethanol Extracts of Lavandula Coronopifolia Against Human Breast Carcinoma Cell line (MDA-MB-321)

Background: Breast cancer is the most common cause of deaths in women. The search for traditionally used medicinal plants which can serve as non-toxic and affordable anticancer drugs is the need of the hour. This study aimed to investigate the anticancer potential of extracts of L. coronopifolia against human breast carcinoma cell line (MDA-MB-321). Methods: The MDA-MB-231 cells were plated in 96 well plates and exposed to 10-1,000 µg/ml of L. coronopifolia for 24 h. The cytotoxic response of different extracts was measured by MTT assay, neutral red uptake (NRU) assay and cellular morphological alterations under the microscope. Results: A concentration-dependent decrease in the cell viability of MDA-MB-231 cells was observed after the exposure of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia. The cell viability was found to be 82%, 89% and 98% at 1000, 500 and 250 µg/ml, respectively in petroleum ether, 37%, 75% and 88% at 1,000, 500 and 250 µg/ml, respectively in ethyl acetate extract, 30%, 35% and 64% at 1,000, 500 and 250 µg/ml, respectively in chloroform extract and 44%, 65% and 82% at 1000, 500 and 250 µg/ml, respectively in ethanolic extract of L. coronopifolia exposed MDA-MB-231 cells. The results also exhibited morphological alterations in MDA-MB-231 cells exposed to various extracts. The cells treated with 250- 1000 µg/ml lost their original morphology and cell linkage as compared to control cells. Conclusion: These preliminary results suggest the promising anticancer potential of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia against MDA-MB-321 cells. Further studies are required to know the mechanism(s) involved in the cell death.


Introduction
The cancer has been recognized as second foremost cause of death worldwide after cardiovascular diseases (WHO, 2015). Among women, breast cancer is the most common type of cancer and the major cause of death (Esmailpoor et al., 2019). In developing countries, breast cancer is prominent reason of death and in developed countries, breast cancer is second leading cause of death (Rafieian-Kopaie and Nasri, 2015). In spite of new diagnosis techniques such as mammography and needle sampling, no significant change has been observed in the mortality rate since many decades. The increasing tumor heterogeneity, drug resistance and high cost of available therapeutic methodologies are present concern that are connected with effective treatment of breast cancer (Chakraborty and Rahman, 2012). Although, excessive progresses have been made to understand the pathophysiology of disease and the development towards conventional chemotherapy even though there is still shrubs and herbaceous plants. Lavandula plants are known for their antioxidant, insecticidal, anti-inflammatory, sedative and spasmodic properties (Wichtl, 1994). L. augustifolia exhibited anti-inflammatory and analgesic activities (Valiollah et al., 2003). L. stoechas displayed hepato-and neuroprotective properties (Slimen et al., 2015). L. pubescens is known for possessing a broad spectrum antimicrobial (Gouda, 2017), anti-inflammatory and hepatoprotective activities (Mousa et al., 2018). L. coronopifolia Poir, L. dentata and L. pubescens Decne are the three species found in Saudi Arabia. These plants have been used in traditional system of medicine. The infusion of flowers of L. dentata is used for urine retention and removal of stones from kidney and ureter. Similarly L. pubescens is known to relieve headache and cold (Rahman, 2004). The literature survey also reveals L. coronopifolia possess antioxidant (Abeer, 2011), antibacterial (Talibi, 2011) and hepatoprotective potential (Farshori et al., 2015). However, the anticancer properties of L. coronopifolia have not been reported so far. Hence, this study was designed to explore the anti-cancer effect of various solvent extracts of L. coronopifolia on human breast carcinoma cell line (MDA-MB-321).

Chemicals and consumables
Cell culture medium (DMEM), Fetal bovine serum, and trypsin-EDTA (0.25%) were procured from Gibco, Invitrogen, USA. Cell culture plates and other plastic wares used in present study were purchased from Nunc. MTT, neutral red dye, other specified chemicals and solvents were obtained from Sigma.

Plant material and extractions
The plant used in present investigation was collected from Shaza Mountains in Saudi Arabia. The plant was authenticated by a taxonomist and a voucher (# 15799) was submitted at the herbarium, Department of Pharmacognosy, College of Pharmacy, King Saud University. The aerial parts of Lavandula coronopifolia plant were air dried and grounded to a coarse powder. For the extraction, powdered plant material was macerated in petroleum ether, ethyl acetate, chloroform, and ethanol successively. The procedure was repeated several times. At each stage the filtrate was collected and the solvent was then evaporated to obtain the extracts of respective solvents.

Cell culture
MDA-MB-231, a human breast cancer cell line was originally purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The cell line, MDA-MB-231 was grown in DMEM medium with 10% fetal bovine serum, 0.2% NaHCO3 and 1% antibiotic/antimycotic solution in a CO 2 incubator at 37 o C in high humid atmosphere.

Experimental design
MDA-MB-231 cell line was exposed to 10 -1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After the treatment, cytotoxicity assessment of L. coronopifolia extracts was done using MTT assay, neutral red uptake assay, and morphological assessments on MDA-MB-231 cell line.

Cytotoxicity Assessment of L. coronopifolia extracts (MTT assay)
Cytotoxic effects of L. coronopifolia extracts was performed using MTT assay as described by Mossman et al., 1983. In brief, MDA-MB-231 cells (10,000 in numbers) were seeded in 96 well culture plates and allowed to adhere in CO 2 incubator for overnight. Then, MDA-MB-213 cells were treated with 10 -1,000 µg/ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After exposure, 10 μl/well MTT solution (5 mg/ml) was added and incubated further for 4 h. Supernatant was removed from the wells and DMSO (200 µl) was added in each well and mixed. The absorbance of plate was then read at 550 nm wave length.

Cytotoxicity Assessment of L. coronopifolia extracts (NRU assay)
Cytotoxic effects of L. coronopifolia extracts was also assessed by NRU assay as described by Borenfreund and Puerner, 1984. In brief, MDA-MB-231 cells (10,000 in numbers) were seeded in 96 well culture plates and allowed to adhere in CO 2 incubator for overnight. Then, cells were exposed to various concentrations (10 -1,000 µg/ml) of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia for 24 h. After the treatment, neutral red (50 μg/ml) was added in wells and further incubated for 3 h. Then, solution was rapidly washed with solution (0.5% formaldehyde and 1% calcium chloride) and the dye was extracted in a solution of 1% acetic acid and 50% ethanol. The absorbance of plate was then read at 550 nm wave length.

Morphological analysis
For the analysis of morphological assessment, the MDA-MB-231 cell line was exposed to 10 -1,000 µg/ ml of petroleum ether, ethyl acetate, chloroform, and ethanol extracts of Lavandula coronopifolia for 24 h. After the treatment with Lavandula coronopifolia, cell images of MDA-MB-231 cells were grabbed under the phase contrast inverted microscope at 20× magnification.

Statistical analysis
The p values <0.05 were taken as statistically significant between control and treated groups using One-way ANOVA. The results are expresses as Mean + SD from three independent experiments.

Discussion
Breast cancer is one of the vigorous problems and major cause of mortality in female globally. Therefore, the aim of this study was to investigate anticancer potential of L. coronopifolia extract against human breast carcinoma (  cell line in this study was chosen, because it has been proven as a good in vitro model system to investigate the cytotoxicity and anticancer potential of various plant extracts against breast cancer (Alshatwi, 2011;Dilshad et al., 2012, Ghafari et al., 2017Nasr et al., 2018). Traditionally medicinal plants have been proven to play an important role in the treatment of various cancer diseases (Bournine et al., 2017). Numerous researches have been shown beneficial effects of plant extracts against various cancer cell lines (Pan and Ho, 2008;Farshori et al., 2013. It is well documented that plants and their components could act as cytotoxic/antiproliferative effects and induced apoptosis in various cancer cell lines Al-Oqail et al., 2013). These days, the research on natural/plant phytochemicals are increasing actively because of their potential health benefits and less side effects. In this study, we have chosen Lavandula coronopifolia plant because it is reported that L. coronopifolia possess antioxidant (Abeer, 2011), antibacterial (Talibi, 2011) and hepatoprotective potential against ethanol induced oxidative stress and cytotoxicity (Farshori et al., 2015). But anticancer properties of Lavandula coronopifolia have not been reported against human breast carcinoma cells. The cytotoxic effects of potential of different extracts of L. coronopifolia has been assessed using various parameters, such as MTT assay, NRU assay and morphological alterations in MDA-MB-231 cell line treated with different concentrations for 24 h. The MTT and NRU assays showed that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia decreases the cell viability of MDA-MB-231 cell line in a concentration dependent manner.
The chloroform extract was found to be more cytotoxic towards MDA-MB231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. The cytotoxicity of ethyl ether, chloroform and ethanolic extracts were found at 250 µg/ml and above concentrations of Lavandula coronopifolia, whereas, 100 µg/ml and lower concentrations of Lavandula coronopifolia did not show significant cytotoxicity to MDA-MB-231 cells. The concentrations range selected in study was chosen from previously reported literature, since exposure of different natural products have been studied by different investigators under in vitro conditions (Maria et al., 1997;Nguta et al., 2012;Solanki and Selvanayagam, 2013). The results of the present study are in accordance to previous report on cytotoxic potential of different plant extracts (Kaneshiro et al., 2005;Kumar et al., 2011). The ethanolic extract of Nigella sativa plant have also been reported to possess cytotoxic effects against P388, HepG2, Molt4 and Lewis lung carcinoma cell lines (Swamy and Tan, 2000). The ethyl acetate extract of Bismillah leaf (Vernonia amygdalina) induced cytotoxicity and morphological alterations have also been reported against human glioblastoma cell line (U-87) (Bin Rohin et al., 2017). In other study, the cytotoxic/antiprolifertaive potential of ethyl acetate extract against HT-29 human colon cancer and MCF-7 human breast cancer cells have also been reported (Lansky and Newman, 2007). The anticancer potential of chloroform extract and sub-fractions of Nepeta deflersiana against MCF-7 human breast cancer and A549 human lung carcinoma cell lines have been studied . Recently, Usmani et al., (2018) have also reported the antiproliferative and cellular oxidative stress induced by Cordia dichotoma (Linn.) extract and its fractions on human cervix epitheloid (HeLa) and human lung (A549) carcinoma cells. The anticancer/cytotoxic effects of L. coronopifolia extracts in this investigation suggest that the cytotoxic response of different extracts obtained could be due to the presence of active components, which is showing the anticancer activity of L. coronopifolia extracts against human breast cancer cells MDA-MB-231.
In conclusion, the results from present investigation demonstrated that petroleum ether, ethyl acetate, chloroform, and ethanol extracts of L. coronopifolia decreased the cell viability and altered the cell morphology of human breast cancer cell line (MDA-MB-321). The results also showed that chloroform extract was found to be more cytotoxic towards MDA-MB-231 followed by ethyl acetate extract and ethanolic extract. Petroleum ether extract was found to be less cytotoxic as compared to other extracts. Further, more studies are required to know the mechanism(s) of cell death involved in this process.