1.      Introduction

Cancer is one of the life threatening and major health issues across the globe. According to a statistic, cancer is accounted for 11.4 million new cases and 7.4 million deaths in 2004.Cancer incidence in South East Asia region was 1.7 million in 2004 (The global burden of disease 2004 update). Cancer has become one of the ten leading causes of death in India [1]. Conventional methods of cancer treatment hold several undesirable side effects and hence an alternative source for obtaining anti-cancer drugs are must require. The promising sources of these alternative drugs are from plants [2]. Few reports were available on the usage of this marine halophyte as food [3,4], source of soluble polysaccharides [5] and anti-cancer drug source [6-9]. Hence, we decided to exploit this plant against the hunt for anti-cancer drugs.

2.      Materials and Methods

4.1. Study Area Description

The present study was carried out in the marine environment of South east coastal areas of Thanjavur and pudhukottai district. The sampling spots are located on 10.20^°N 79.24^°E on the coast of the Bay of Bengal, South East coast of Tamilnadu. The sampling locations included are Adirampattinam, Rajamadam and Mallipattinam (Thanjavur district, Tamilnadu) and Memesal (Pudhukottai District) (Figure 1).

Adirampattinamis located on 10.20^°N 79.24^°E on the coast of the Bay of Bengal. Being close to the equator, Rajamadam is a small village located at the longitude of 79.34^°N and latitude of 10.30^°E. Mallipattinam is a small village in Thanjavur District in Tamilnadu State. Located on Lat. 10.27^°N and Long79.31^°E, Memesal is one of the Villages in Pudhukottai District in Tamilnadu. Located on Lat. 9 .56° N and Long. 79 .10^°E.

4.2. Collection and Processing of plant

The fresh plant was collected from south east coast of Tamilnadu. The plant was identified by Dr. V. Ramasubramanian, Head, Department of Botany (UG), Ayya Nadar Janaki Ammal College, (Autonomous), Sivakasi. Later the plant was washed in running tap water chopped and kept shade dried for 20-25days in a dark place.

4.3. Screening of Phytochemicals

The different analytical tests were performed in order to establish the chemical profile of the extracts. The following chemical tests like Test for Flavonoids [10], Tannins [10], Saponin [10], Steroids [10], Quinones [10], Coumarins [10], Terpenoids [10], Cardiac glycosides [10] and Phlobatannins [10] were performed.

4.4. Chemical fingerprint of S. europaea L. by GC-MS

The chemical fingerprint of the halophyte was obtained through GC-MS analysis.Model No: Agilent 7890B Gas Chromatograph connected with 5977A MSD mass spectrometry. The Gas chromatography unit was equipped and coupled to a HP_5MS 5% Phenyl Methyl Silox -60^°C-325^°C (325^°C) column and its dimension of the column is 30m×250µm×0.25µm. Prior to the initiation of the phytochemical scanning procedure the instrument was set to an initial temperature of 50^ºC. At the end of this preparatory period the oven temperature was rose up to 300^ºC, at the rate of an increase of 5^ºC/min, and maintained for 9 min. Following are the parameters need to be ensured for the successful operation of the instrument. The temperature of the sample injection port was ensured as 250^ºC and the flow rate of Helium was ensured as 1ml/min. The ionization voltage was set at 70eV. The extract samples were injected in split mode. Scan range was set at 45-450 (m/z). The structure of the phytoconstituents was retrieved using computer searches on a NIST Version-Year 2011.

4.5. Evaluation of Anticancer activity of Phytocompounds by MTT assay

4.5.1. Preparation ofCell line

The cell line used in the present study was Human breast adenocarcinoma cell lines (MCF7) and it was obtained from National Centre for Cell Science (NCCS), Pune. The cell line was maintained in Eagles Minimum Essential Medium containing 10% Fetal Bovine Serum (FBS). The cells were maintained under the controlled incubatory conditions like the temperature was at 37^ºC, 5% CO₂, 95% air and 100% relative humidity.To maintain the viability of cells in the cell line it was and passaged weeklyaccording to Mosmann [11].

4.5.2. Cell treatment procedure

According to Monks [12], after monolayer cells were formed it was detached with enzyme based salt. The procedure was carried out to make detached single cell suspensions from monolayer and viable cells were counted using a hemocytometer. The cells were diluted with medium containing 5% fetal bovine serum to yield a final concentration of cells and it was calculated to be 1x10⁵ cells/ml. Initially about one hundred microliters of cells were pipetted and transferred per well in 96-well plates and the satisfied plating density was calculated to be 10,000 cells/well.To promote the cell attachment certain parameters were adapted like 37^°C, 5% CO₂, 95% air and 100% relative humidity. Exactly after 24 h of incubation the cells grown the 96-well plates were treated with different concentrations of the test samples. The plant extract was dissolved in Dimethyl Sulfoxide (DMSO). Once it was dissolved an aliquot of the test sample solution was further diluted to obtain a desired final maximum concentration with serum free medium.Using a microliter pipette 100 µl of these different sample dilutions were transferred to the appropriate wells which were already loaded with 100 µl of medium. Following the addition of test sample, the microtitre plates were subjected to incubation for 48 h with the following specified parameters viz 37^°C, 5% CO₂, 95% air and 100% relative humidity. To compare the test sample equally another preparation was maintained but that was free from test sample.

4.5.3. MTT Assay

The MTT assay was performed according to Monks [12]. After 48 h of incubation, 15µl of MTT (5mg/ml) in Phosphate Buffered Saline (PBS) was added to each well and incubated at 37^ºC for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The % cell inhibition was determined using the following formula.

% Cell Inhibition = 100 - Abs (sample)/Abs (control) x100

To understand the relationship between % Cell inhibition and Log Concentration Nonlinear regression graph was plotted and IC₅₀ was determined using Graph Pad Prism software.

3.       Results and Discussion

5.1. Identification of the Plant

The halophyte was identified as Salicornia europaea L (Figure 2).

. Extraction of Phytochemicals by Soxhlet

The dried biomass of the halophyte was extracted with methanol and ethyl acetate (Figure 3) under controlled environment.

The ethyl acetate extract of the biomass was pale yellow and methanol extract was dark brown respectively. The optical property of the extract was bit critical that the ethyl acetate extract was transparent whereas the methanol extract was opaque (Figure 4).

Screening of Phytochemicals

Qualitative analysis of the ethyl acetate and methanol extracts were carried out. The study revealed the presence of two important phytochemicals called Tannins and Coumarins in methanol extract and flavonoids in ethyl acetate extract (Table 1). Essaidiet al. (2013) [13] reported the presence of phenonic acids and flavonoids. The study slightly correlated with above finding further it becomes very clear that the lack of phenonic acids might be due to the condition in which the halophytes are growing. In a detailed study conducted by Cybulskaet al. 2014 [14] had confirmed fatty acids, terpenoids, flavonoids, alkaloids, steroids, tannins, saponins, quinones and coumarins. It was found that many of the constituents reported to have therapeutic values in humans. In the present study, phytochemical screening had paved the way for the confirmation of same type of phytochemicals in the halophyte thus it leads to the confirmation that there is no connectivity between phytochemicals and geographical distribution

Chemical fingerprint of S.europaeaL. by GC-MS

The list of phytochemicals present in both extracts wasscanned through GC- MS and the results were tabulated (Table 2 & Table 3). The analysis had confirmed the prolific nature of compounds in the selected halophyte. Further the GC-MS analysis confirmed the presence of structurally diversified compounds based on retention time (Tables 4& Table 5). The detailed reports on the chromatograms are given in Figures 5 & 6 respectively.

The present study was correlated to the report given by Iscaet al. 2014 [15], Zhaoet al. 2014 [16] had also confirmed the presence of triterpenes and saponins in the solvent extract of the halophyte. Similarly, in a study carried out by Samuelet al. 2016 [9] reported that this halophyte is the source of prolific chemicals which is further confirmed in the present study. The study correlates with Das Neves Costaet al. 2015 [17] who had reported the selection of solvent system plays a critical step in the extraction procedure. Radwan [18] had studied the phytochemical profile of S. fruticosa L. In the report he had unveiled that there are 23 compounds by GLC but in the present study fractionated 32 and 29 compounds respectively. Thus it gave further evidence that Salicornia europaea L. is a source of prolific and structurally diversified chemicals.

5.5. Cytotoxic activity of Phyto chemicals

The concentrated crude sample obtained from ethyl acetate and methanol was subjected to evaluate the anti-cancer activity. Before the analysis was begun the crude samples was diluted and taken in the following concentration 12.5µg, 25µg, 50 µg, 100µg and 200µg (Tables 2 and 3).

Both the extracts were evaluated for anti-cancer potential. The cytotoxicity pattern of methanol extract and its corresponding nonlinear graph is illustrated in (Figures 7 and 8) respectively. The graph revealed the significance of relationship between dosage concentration and cytotoxicity.

It was observed that the ethyl acetate extract had significant cell inhibitory activity against Human breast adenocarcinoma cell line (MCF7). Maximum cell inhibition was observed in 200µg of the compound tested prepared from ethyl acetate and methanol. The percentage of cell population inhibited at 200 µg was 81.71% (ethyl acetate) and 79.49% (methanol). Further the IC₅₀ value of the compound tested against the cell line 97.9µg/ml (ethyl acetate) and 117.1µg/ml (methanol). The cytotoxicity pattern of ethyl acetate extract and its corresponding nonlinear graph is illustrated in [Figures 9&10] respectively. The graph revealed the significance of relationship between dosage concentration and cytotoxicity. Lellau and Liebezeit [19] studied the cytotoxic activity of salt marsh Daphnia sp. Its activity was comparatively lesser than Salicornia europaea L. The present investigation on the halophyte reinforced that it is advantageous. The study also related to Essaidi [13] et al. 2013 in the aspect that the methanolic extract has cytotoxic activity but at the same time cytotoxic activity of the methanolic fraction was comparatively lesser than ethyl acetate extract.

4.      Conclusion

The studies conclude that Salicornia europaea L. is abundant along the Adirampattinam coastal environs of Tamilnadu. Qualitative screening had led us to confirm the presence of Tannins and Coumarins in the methanolic extract and flavonids in ethyl acetate extract. Extensive phytochemical screening was carried out using GC-MS. The study yields the presence of 32 and 29 compounds in corresponding ethyl acetate and methanol extracts respectively. The ethyl acetate fraction of this halophyte had exhibited a maximum cell inhibition when it was added to the cell line in 200µg. Further the IC₅₀ value also determined for the same. The value was comparatively less with that of methanolic extract.

5.      Acknowledgement

The author expresses his thanksand gratitude to The Management and The Principal of Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi for providing laboratory support with all needed chemicals and instruments for the successful completion of the work analysis and anti-cancer studies. 

Figure 1: Sampling spots along the south-east coast of Tamil Nadu.

Figure 2: Salicornia europaea L. grown in large scale along the south-east coast of Tamil Nadu.

Figure 3: Phytochemical extraction of Salicornia europaea L.

Figure 4: Methanol and ethyl acetate extract of S. europaea L.

Figure 5: GC-MS chromatogram of Methanol extract of Salicornia europaea.

Figure 6: GC-MS analysis of ethyl acetate extract of Salicornia europaea.

Figure 7: Cytotoxicity pattern of Methanol extract of Salicornia europaea.

Figure 8: Nonlinear graph showing the relationship between doses of phytocompounds (Methanol) % of cell inhibition.

Figure 9: Cytotoxicity pattern of Ethyl acetate extract of Salicornia europaea.

Figure 10: Nonlinear graph showing the relationship between doses of phytocompounds (Ethyl acetate) % of cell inhibition.

Table 1: Screening of phytochemicals in different Extract.

Table 2: List of phytochemicals present in ethyl acetate extract.

Table 3: List of compounds present in the GC-MS analysis of Methanol extract.

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