Anticancer potentials of leaf, stem, and root extracts of Achyranthes aspera L

Achyranthes aspera L. (Amaranthaceae), an herbaceous roadside weed in various parts of India, has several therapeutic uses, including the treatment against cancer. This investigation was undertaken to identify the bioactive compounds conferring cytotoxicity to the extracts from its biomass. The powdered leaf, stem, and root biomass was extracted separately in ethyl acetate, acetone, ethanol, and methanol. Each extract was tested against the HeLa cell line for the cytotoxicity, but the root-acetone extract was the most cytotoxic. This extract revealed eleven bands. The solution obtained from the ninth band (Rf = 0.87±0.06) exhibited more than 90% inhibition of HeLa cells. The LCMS analysis of this solution showed the presence of 37 compounds, out of which few compounds had been reported from different plants to possess cytotoxicity in various systems.


Introduction
Cancer is one of the most severe global health concerns in developing and developed countries alike. The drugs and therapies used in cancer treatment have several harmful side effects. Therefore, it is essential to search for plant-based natural anticancer agents that are safe and equally efficient. Naturally occurring phytochemicals from different medicinal plants contain a wide range of secondary metabolites with curative properties (Solowey et al., 2014;Greenwell and Rahman, 2015). About 60% of currently used chemotherapeutic drugs are isolated from natural products of more than 3000 plants worldwide (Solowey et al., 2014).
Achyranthes aspera L. (Amaranthaceae) is a herbaceous roadside weed that grows in various parts of India (Singh et al., 2011). Different parts of the plant, as well as the whole plant, have many therapeutic uses.
Various extracts prepared from A. aspera have been shown to possess in vivo cytotoxic activity (Subbarayan et al., 2012) including killing human colon cancer cells  by triggering the mitochondrial apoptosis pathway and s phase cell cycle arrest (Arora and Tandon, 2014). However, there is meagre literature on the bioactive compounds conferring cytotoxicity to these extracts. Therefore, the present investigation was undertaken to identify the potential anticancer bioactive molecules from A. aspera against HeLa (human cervical carcinoma) cell line.
Collection of plant material The healthy plants of A. aspera were collected in July and August from the campus of Fergusson College, and the Savitribai Phule Pune University, Pune (accession no. MCASC-BOTLAB-2017-1). The identification of the collected plant was authenticated from the Botanical Survey of India, Western Circle, Pune. The leaves, stem, and roots were separated, washed thoroughly in running tap water, blotted dry, and cut into small pieces. The fresh biomass was layered on sheets of filter paper and dried in the shade at ambient temperature for about one week. The dried biomass was powdered in a mechanical grinder, and the powders were stored at -20 °C in polythene bags with airtight zip lock.

Extraction of phytochemicals
The phytochemicals from the powdered leaf (L), stem (S), and root (R) biomass were extracted in a Soxhlet extractor separately in ethyl acetate (EA), acetone (Ac), ethanol (Et), and methanol (Me). Twentygram biomass was wrapped in Whatman No. 1 filter paper and extracted for five h in 180 mL solvent. The extract was suction filtered through a Whatman No. 1 filter paper disc placed in the Buchner funnel. The clear extract was evaporated to dryness in a 250 mL beaker placed in a hot water bath. The mass of dried residue was measured on an electronic weighing balance (CAS-250, Contech Instruments Ltd., India). The residue was suspended in 5.0 mL of solvent for eight h, after which it was centrifuged at 5000 × g. The clear supernatant was collected in glass vials and stored at -20 °C until further use. Thus, four types of leaf extract L-EA, L-Ac, L-Et, L-Me; four types of stem extracts S-EA, S-Ac, S-Et, and S-Me; and four types of root extracts R-EA, R-Ac, R-Et, and R-Me were prepared. Each extract was diluted with the solvent used for extraction to yield a working solution with concertation of 500 micrograms per milliliter (µg/mL).
Cell lines and cell culture In vitro cytotoxic effects of all twelve extracts made from A. aspera leaves, stem and roots were studied on human cervical adenocarcinoma (HeLa) cell line. The HeLa cell line was procured from National Centre for Cell Sciences (NCCS) Pune, India. The cell lines were maintained in T-25 flasks containing MEM essential media along with 10% heat-inactivated fetal bovine serum (FBS) and Penicillin-Streptomycin at 100 U/mL and 100 μg/mL, respectively. Cells were maintained under an atmosphere of 5% CO2 and 95% humidity at 37 °C.
In vitro cytotoxicity assay In vitro cytotoxicity of each extract on HeLa cells was determined by using MTT assay. The cells were maintained in MEM medium supplemented with 10% FBS, 100 U/mL Penicillin, and 100 μg/mL Streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. Ten thousand cells in the respective medium were seeded in a 96 well plate and incubated at 37 °C in the atmosphere of 5% CO2. After 24 h incubation, the confluent cells were exposed to the respective treatment of each of the 12 extracts at the concentrations of 0, 25, 50, 100, 200, and 400 µg/mL in culture medium without FBS and incubated for further 48 h at 37 °C and 5% CO2 atmosphere. After 24 and 48 h of treatments, cells were washed with sterile phosphate-buffered saline, and the cell proliferation was determined using MTT dye. Ten µl MTT (5 mg/mL) was added to each well, and the plates were further incubated at 37 °C in a 5% CO2 atmosphere. After four h incubation, the MTT solution was carefully removed, and 200 μL of DMSO was added to each well. The absorbance of purple color developed was measured at 560 nm using a UV-Vis spectrophotometer (Model 1800, Shimadzu, Japan). The results were expressed as a percentage of cell inhibition as compared to the control.
The extract showing the highest cytotoxicity was subjected to TLC separation, and the solution made from each eluted TLC band was checked for cytotoxicity at the concentrations of 0, 2.5, 5, 10, 25, 50, and 100 µL/mL against the HeLa cells as described earlier.

Cell morphology
Cancer cells (1x10 6 cells/well) were seeded in a six-well culture plate and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. Later, the cells were treated with 0, 25, 50, 100, 200, and 400 µg/mL concentrations of acetone root extract of A. aspera in respective culture medium without FBS and further incubated for 48 h.
The morphology of the cells was then examined under an inverted phase-contrast microscope (Primovert, Carl Zeiss).

Separation of the extract by using Thin Layer Chromatography (TLC)
The extract was subjected to TLC for the separation of phytochemicals. The pre-coated silica plates (TLC Silica Gel 60 F254, Merk, India) were used for the separation. Analytical separation of extract containing 25 µg dissolved residue was carried out in various solvent systems differing in polarity. A preparative TLC plate of 200 mm × 200 mm was used to separate 350 µg dissolved residue in the best solvent system (20% acetone in ethyl acetate) identified from the separation on analytical plates. Two such plates were simultaneously developed in one chromatography chamber. At the end of separation, the solvent front was marked, and the Rf value of each visible band was calculated.

Elution of separated bands
The TLC plate was air-dried, and each band was scraped off from the plate by using a scalpel, and the powder of two identical bands from two plates was pooled and suspended in 500 µL of acetone for eight h. The suspension was then centrifuged to remove the silica powder, and the clear supernatant was stored in glass vials. The solution thus prepared from each band was subjected to cytotoxicity testing on a v/v basis (2.5 to 100 µL/mL) against the HeLa cell line. The solution showing the highest percent inhibition was subjected to LC-MS analysis.

LC-MS analysis
LC-MS profiling of TLC band that showed the highest cytotoxic activity was performed on Agilent G6540B Quadrupole Time of Flight (QTOF) binary LC system equipped with Dual Agilent Jet Stream Electrospray Ionization (AJS ESI) and separated on Agilent Zorbax column (50 × 2.1 mm, 1.8 μm) using aqueous 0.1% formic acid (Solvent A) and acetonitrile (Solvent B). A stepwise increasing polarity gradient of solvent A and solvent B was applied at the flow rate of 0.3 mL/min for 30 minutes. The capillary voltage, cone voltage, and fragmentor voltage were 3.5 kV, 45 V, and 150 V, respectively. The gas temperature was set to 325 °C. Electroscopy mass spectra data were recorded in a positive ionization mode. The data was acquired at a scan rate of 2 spectra/sec in the mass range of 60-1700 m/z and analysed with Mass Hunter Qualitative Software and METLIN database. The class of the putatively identified compounds was deduced from the websites of the metabolomic workbench (www.metabolomicsworkbench.org) and the Lipidomics Gateway (www.lipidmaps.org).

Statistical analysis
All experiments were performed in three replicates, and the values were expressed as mean ± SD or mean ± standard error (SE) of means.

Results
Isolation and characterization of phytoconstituents from different parts of A. aspera The result of the yield of extractable constituents is presented in Table 1. The percentage yield of extractable constituents was different in different solvents as well as source biomass. The stem biomass gave higher yield in all the solvents used except ethyl acetate, which gave the highest yield from leaf biomass. In vitro cytotoxicity of A. aspera extracts To analyse the in vitro cytotoxic effects of ethyl acetate, acetone, ethanol and methanol extracts of root, stem, and leaf biomass of A. aspera, cell viability, and cell proliferation was determined by using MTT assay. The cytotoxic activity of these extracts was tested in vitro at the concentrations between 25 -400 μg/mL against the cervical adenocarcinoma cell line (HeLa) exposed to the extracts for 24 h and 48 h. It was observed that the acetone and ethanol extracts of root biomass exhibited profound cytotoxic activities in a dose-dependent manner. In contrast, all other extracts of leaf and stem biomass showed comparatively lower cell growth inhibition even at higher concentrations.
With a more prolonged exposure for 48 h, all the extracts showed varying degrees of increase in the cytotoxicity ( Figures 1E to 1H). At the highest concentration of 400 μg/mL, the minimum increment of 4.65% was observed from R-Ac extract ( Figure 1F) whereas a maximum of 68.66% increment was shown by L-Et extract ( Figure 1G).
Thus, the R-Ac extract of A. aspera exhibited the maximum cytotoxic activity against HeLa cells after 24 h exposure. Morphological features of treated cells observed under an inverted phase-contrast microscope revealed distinct cellular morphological alterations such as loss of membrane integrity. Such cells, therefore, appeared shrunken and had cytoplasmic condensation indicating unhealthy nature. On the contrary, the untreated control cells appeared normal. These results indicate that the cells treated with higher concentrations of R-Ac extract had died, whereas untreated cells remained alive. The R-Ac extract subjected to TLC separation showed 11 distinct bands. The Rf value and the cytotoxicity potential of the solutions constituted from each band are presented in Table 2 and Figures 2A and 2B.
Solutions prepared from all eleven bands separated on a TLC plate showed cytotoxicity against HeLa cells. The cell growth inhibition ranged from about 29% to 86% after 24 h exposure to the solutions. There was not much difference between cell toxicity observed after 24 h and 48 h treatments. The maximum difference (∼14-15%) was observed in the cells treated with the solutions prepared from the 1 st and the 11 th bands. In contrast, a minimum of 0.9% difference was observed from the solution of the second band. The 9 th band solution showed the maximum cytotoxicity against HeLa cells. The acetone solution prepared from the 9 th band when subjected to LC-MS separation revealed 37 putatively identified compounds with match scores higher than 90 (Table 3).
LC-MS analysis of the phytochemicals from the solution prepared from the 9 th band on the TLC plate thus reveals its heterogeneous nature. It contained 32% lipids, 11% organic acids, 7.9% benzenoids, 2% sterols, and 2.6% each of terpenes, quinones, and alkaloids.

Discussion
In the present investigation, HeLa cells treated with various extracts for 24 h and 48 h suffered dosedependent cytotoxicity (25-400 µg/mL) coupled with distinct morphological damage including cellular and nuclear shrinkage. It was observed that the cancer cells died due to reduced cell proliferation when exposed to extracts prepared from biomass of A. aspera, and the extract of roots in acetone had the maximum cytotoxicity against HeLa cancer cell line. Therefore, it can be concluded that at least a few of the 37 compounds identified by the LC-MS analysis of the root-acetone extract possess anticancer property. For example, the polyphenols from A. aspera (100 mg/kg, p.o.) were shown to possess synergistic anti-cancerous and cytokine based immunomodulatory role in mice suffering from urethane-induced lung cancer (Narayan and Kumar, 2014). The LC-MS has detected eupatorin (C18H16O7, RT=12.4), a flavonoid polyphenol in the 9 th TLC band of R-Ac extract. It has shown an anti-proliferative activity against several cancer cell lines including human uterus carcinoma (HeLa), human gastric adenocarcinoma (MK-1), and murine melanoma (B16F10) (Nagao et al., 2002); liver murine colon carcinoma (26-L5) (Tezuka et al., 2000), and human breast cancer cell line (MDA-MB-468) (Androutsopoulos et al., 2008).

Conclusions
The present investigation supports the claims of anticancer properties attributed to Achyranthes aspera L. The LC-MS analysis of the acetone extract of its roots has revealed the presence of a few anticancer compounds reported in the literature. The global increase in the number of cancer patients, limitations of conventional therapies, the toxicity of drugs used in cancer treatment, and expensive and time consuming traditional anticancer drug discovery approaches have compelled the researchers to look for inexpensive and efficient alternatives in the kingdom of plants. Medicinal plants like A. aspera are natural reservoirs of bioactive phytochemicals that are effective against a wide range of diseases, including cancer. Moreover, they can be incorporated as complementary treatments alongside conventional drugs. Their use in managing or restraining the cancerous growth can be a better alternative to the conventional allopathic line of treatment on various cancers. Several plant-derived biomolecules like alkaloids, flavonoids, terpenes, and other secondary metabolites, have been authorized and subscribed as anticancer medicines. The notable examples that have been through clinical trials are taxol from Taxus brevifolia Nutt., vinca alkaloids from Catharanthus roseus G. Don., curcumin from Curcuma longa L., podophyllotoxin from Podophyllum emodi Wall., and camptothecinderived topotecan from Camptotheca acuminata Decne. Their use as eco-friendly, biocompatible, and costeffective alternative in the treatment of various types of cancers is gaining momentum worldwide. There is a genuine need for many such efficient anticancer drugs with little or no side effects. At the same time, it is necessary to evaluate such anticancer biomolecules for their effectiveness, possible applications, and toxicological studies under in vitro conditions and in the clinical trials as well.