Annihilating efficacy of Euphorbia hirta L. extracts on Artemia salina Nauplii and human breast adenocarcinoma cells (MCF-7 & MDA-MB-231)

Cancer is a leading cause of death both in developed and developing countries. Recently the secondary metabolites produced by plants are being investigated due to their promising anticancer activities. Accordingly, in the current study the anti-cancer potentials of Euphorbia hirta L., a renowned medicinal plant was explored for its anticancer activity. The methanol and aqueous extracts of Euphorbia hirta L. (EHA and EHM) were tested for their toxicity against Artemia salina nauplii, and for cytotoxic potentials in MDA-MB-231 and MCF-7 human breast adenocarcinoma cell lines. Both the extracts EHA and EHM showed maximum toxicity towards Artemia salina among which the methanol extract was able to annihilate all the nauplii in its highest concentration. Excitingly, Euphorbia hirta L. extracts exhibited potent cytotoxicity selectively against human breast cancer cells (MDA-MB-231 and MCF-7) than in normal cells (VERO). In conclusion, the results suggest that EHM extract of the selected plant might possess beneficial ingredients to treat human breast cancers especially against ER-positive breast cancer.


INTRODUCTION
Cancer is well-known as a multifactorial disease due to abnormal proliferation of cells and is one of the major causes of death worldwide. Various genetic and epigenetic events create an accumulative phenomenon that lead to cancer development and progression (Wang et al., 2001). Treatment of cancer is particularly challenging and it inflicts a severe burden on the public health system. Conventional chemotherapy is associated with anticancer drugs with low therapeutic index and results in severe side effects (Khandare et al., 2006). Recent evidences suggest that natural products are good sources for the treatments of human diseases. Medicinal plants have been used to treat human diseases for centuries (Sharma and Moin, 2020). According to the World Health Organisation (WHO) few nations still rely on plant-based treatment as their main source of medicine (Parasuraman et al., 2014). The history dates back to several years in using plants for their medicinal benefits, especially for cancer (Conforti et al., 2008). Chemotherapy seems to be promising where 65% of the drugs used are of natural origin (Cragg & Newman, 2005). Due to the above-said reasons, medicinal plants might prove potential to aid in the development of alternative therapy against cancer.
Euphorbia hirta L. is a very popular herb amongst practitioners of traditional herbal medicine and belongs to the Euphorbiaceae family. Traditionally, aqueous extracts of various E. hirta L. parts were noted to be used to treat various syndromes that include hay asthma, bowel disease, cough, bronchial disease, worm infestation, kidney stones, to decrease lactation, etc. They were also recorded for their benefits as a sedative, anxiolytic, analgesic, antipyretic, and anti-inflammatory agent. Furthermore, studies revealed that E. hirta L. possesses galactogenic, anti-anaphylactic, antimicrobial, antioxidant, anticancer, anti-feedant, antiplatelet aggregation, antiinflammatory, aflatoxin inhibition, antifertility, anthelmintic, anti-plasmodial, anti-amoebic, antimalarial and larvicidal activities (Rajeh et al., 2010). Based on the afore-mentioned literature, the aqueous and methanol extracts of E. hirta L. were investigated to identify potential anticancer properties.

Plant Materials
The fresh plants of E. hirta L. was collected from its natural habitat from Indur village of Dharmapuri district (Tamil Nadu, India). Collected specimens were recorded by maintaining a voucher specimen (GACDPIEH1) in the laboratory for future references. Whole plants were subjected to shade drying and powdered coarsely in a mechanical mixer-grinder.

Preparation of Extracts
Hot extraction was performed in a soxhlet apparatus for the powdered plant material using methanol and water as solvents [sample (g): methanolic/aqueous) = 1:6 ratio]. Obtained extracts were then concentrated in rotavapor R-215 (BUCHI Labortechnik AG, Switzerland) under reduced pressure (72 mbar) at 40 °C to yield dry extracts. Concentrated extracts were then stored in a vacuum desiccator at room temperature until further use. The extracts were named as EHA (Euphorbia hirta L. aqueous) and EHM (Euphorbia hirta L. methanol).

Brine Shrimp Lethality Assay (BSLA)
Toxicity of the plant extracts were evaluated primarily on brine shrimp, Artemia salina nauplii. The lethality assay was performed following the method of Meyer et al. (1982). Briefly, eggs were obtained from STORI Salt Lake Artemia Cysts, Fish Cave Seller, Howrah, WB, India and stored at 4°C in a refrigerator until further use. 1g of the cysts was sprinkled into the glass container (1L capacity) containing sterile artificial sea water (prepared by dissolving 38g of sea salt in 1 litre of sterile water) for hatching. This setup was incubated at room temperature (25-29°C) for 24hrs with strong aeration and continuous light regime. Following which, newly hatched pinkcoloured free-swimming nauplii were harvested from the bottom of the container. Different dilutions of the plant extracts for this assay were prepared by adopting the procedure of McLaughlin et al. (1998). 20mg of the aqueous and methanol plant extracts were separately dissolved in 2ml of suitable solvents (stock solution). These were further diluted to prepare a series of concentrations 1000, 100, 10, and 1ppm (μg/ml).
The assay system was prepared with 4.5ml of sterile seawater containing 0.5ml of the chosen concentration of the plant extracts in watch glass and transferred with 10 nauplii. The setup was allowed to remain for 24hrs, under constant illumination of florescent lamp and the number of survived and dead nauplii were counted and recorded with a hand lens. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation. Experiments were performed in triplicates including solvent and a negative control. Percentage mortality of the extracts was calculated using the formula: % Mortality= No. of dead nauplii/Total No. of nauplii×100. LC 50 values were estimated using a probit regression analysis (Finney, 1971) at 95% confidence intervals.

Cell Cultures and Maintenance
MDA-MB-231 (human breast carcinoma), MCF-7 (human breast carcinoma) and VERO (African Monkey kidney epithelial) cell lines were procured from National Centre for Cell Science (Pune, India). MDA-MB-231 cells were maintained in L-15 (Leibovitz's) culture medium, and MCF-7 and VERO were maintained in Minimum essential medium (MEM) (Eagle) with Non-essential amino acids, all with 10% fetal bovine serum in a humidified atmosphere at 37ºC (with 5% CO 2 for MCF-7 and VERO only). The cell lines were maintained in their growing phase at 70% confluency with regular passaging.

Cytotoxicity Assay
MTT assay was performed as described by Mosmann (1983) with slight modifications. Briefly, a 96-well plate was seeded with a cell concentration of 10,000 cells from column 2-11 and each well were made up to 200μl using respective fresh medium. 200µl of fresh culture medium alone was loaded in control columns 1 and 12. All sides of the plate was sealed and incubated at 37ºC in a humidified atmosphere for 24hrs. After 24hrs, existing medium was aspirated and wells were replenished with 200μl of media containing varying concentrations (12.5, 25, 50, 100, 200μg/ml) of plant extracts and incubated for 24hrs. After incubation, the extract medium was aspirated and fresh medium was added along with 10μl MTT reagent (final concentration of 0.45μg/ml). The plate was then covered with aluminium foil and placed for incubation in a humidified atmosphere for 4 hours at 37ºC. After which, the medium with MTT was removed and 100μl Solubilization solution (DMSO) was added to each well to dissolve formazan crystals. Absorbance was recorded at 570 nm using a Dynex Opsys MR TM Microplate Reader (Dynex Technologies, VA, USA) with a 630nm reference filter. Wells containing cells without extract treatments served as the control. Percentage cytotoxicity was calculated by using the formula:

Where, A c is the Absorbance of control cells and A is the absorbance of the extract treated cells
The IC 50 value was determined by using linear regression equation i.e. Y = Mx+C.
Here, Y = 50, M and C values were derived from the viability graph.

Statistical Analysis
All analyses were carried out in triplicates. For each assay, data were presented as mean±SD from three independent experiments (n = 3). Statistical analyses were performed by Elankanni et al. one-way ANOVA and significant differences between groups were determined at P<0.05. To evaluate relationships between experimental parameters, results were analyzed for correlation and tested for significance by Student's t-test (P<0.05). MATLAB ver. 7.0 (Natick, MA, USA), GraphPad Prism 5.0 (San Diego, CA, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the statistical and graphical evaluations.

RESULTS AND DISCUSSION
Cancer is the most common and deadly diseases in the modern era and in addition, adverse effects of synthetic anticancer drugs are the major limitations in treating cancer. In Asian cultures, medicinal plants are used traditionally to prevent and treat cancers. Medicinal plants have been reported as a good source for the discovery of new chemotherapeutic drugs with high efficiency and fewer side effects (Harvey & Cree, 2010).

Yield of extracts
Extraction is an important step for the detection of bioactive components from plant materials. With the aid of a solvent, desirable constituents are removed from those that are not required from the plant material (Dhanani et al., 2017). In the present study, fifty grams of E. hirta L. whole plant powder upon soxhlet extraction resulted in a yield of 9.2g (percentage extract yield: 18.4% of dry weight) of aqueous (EHA) and 7.3g (percentage extract yield: 14.6% of dry weight) of methanol (EHM) crude extracts. The sample was further stored in a vacuum desiccator until further use.

Brine shrimp lethality bioassay
The brine shrimp lethality bioassay is widely used in the evaluation of toxicity of heavy metals, pesticides, plant extracts, etc. It's a preliminary toxicity screen for further experiments on mammalian animal models (Price et al., 1974;Sorgeloos et al., 1978). The brine shrimp assay has advantages of being rapid (24hrs), inexpensive, and simple. It easily utilizes a large number of organisms for statistical validation and requires no special equipment (McLaughlin et al., 1998). Both methanol and aqueous extracts of E. hirta L. exhibited significant mortality against Artemia nauplii. The percentage mortality for E. hirta L. whole plant aqueous and methanol extracts were depicted in (Table 1 and Table 2). Accordingly, EHA exhibited percentage mortality ranging from 27% to 73% whereas, EHM exhibited 50% to 100% mortality at 1μg/ml to 1000μg/ml concentrations. EHM found to exhibit better mortality in A. nauplii when compared to EHA with an LC 50 of 1±5.4μg/ml than the later 43.25±9.6μg/ml. As mentioned by Meyer (1982), both the plant extracts exhibited an LC 50 value of less than 1000μg/ml which is considered toxic. The methanol extract (EHM) in its highest concentration (1000μg/ml) had killed all the napulii treated.

Selective-cytotoxicity of EHA and EHM towards breast cancer cells
The efficacy of E. hirta L. extracts to inhibit the proliferation of cancer cells were further evaluated using two cancer cells (MDA-MB-231 and MCF-7) and a normal cell (VERO). Both the extracts have exhibited significant cytotoxicity in cancer cells when compared to normal cell (Figure 1 and Figure 2). Similar results were also made by Weisburg et al. (2004), wherein selective destruction of cancer cells was demonstrated. Among the study extracts, EHM had lower IC 50 values of 82.81 and 24.31μg/ml than EHA (92.86 and 53.08μg/ml) in MDA-MB-231 and MCF-7 cells respectively. The extracts EHA and EHM were more effective in controlling the proliferation of MCF-7 cells rather than MDA-MB-231 cells. According to the American National Cancer Institute (NCI), the crude plant extract with an IC 50 value <30μg/ml indicates the promising plant material for further cytotoxic agent purification (Suffness and Pezzuto, 1990). Accordingly, the extracts, EHA and EHM had lower IC 50 values in MCF-7 cells, close and within the range of reported values by NCI when compared to the values of these extracts in MDA-MB-231 cells. Moreover, VERO cells had recorded the maximum Note: *T=Trial (No. of nauplii taken in each trial = 10); There is no probit value for 0% and 100%

CONCLUSION
The study has revealed that the aqueous and methanol extracts of E. hirta L. plant had higher toxicity against the Brine shrimp, A. salina nauplii, where the methanol extract could annihilate all of the napulii in its highest concentration. Both the extracts exhibited higher cytotoxicity in cancer cells (MCF-7 and MDA-MB-231) when compared to normal VERO cells. The results clearly indicate that both the extracts might possess beneficial therapeutic components specifically against ER-positive breast cancer with lesser damage towards normal cells. Hence, these extracts may further be exploited to isolate novel and potential sources of anticancer agents. Note: *T=Trial (No. of nauplii taken in each trial = 10); There is no probit value for 0% and 100