Cytotoxicity studies by MTT Assay
In the present study, the in vitro cytotoxic effects of ethyl acetate extract of Claviceps purpurea on several cell lines was evaluated by micro-culture tetrazolium assay (MTT). MCF-7, HeLa, A549 cell lines and normal cell lines (3T3L1) were evaluated. The cytotoxicity of the fungal extracts was also investigated against the normal Fibroblast cell line. The ethyl acetate extract of Claviceps purpurea showed an exponential increase in cytotoxic activity against both the cells are MCF-7, A549, and HeLa. In A549 cell lines the ethyl acetate extract showed IC50 value is 56.38 µg/mL (Table 1). Standard vinblastine showed 20.73 µM on MCF-7, 24.86 µM on A549 and 21.23 µM on HeLa cellines. For further activity based on IC50 values A549 for ethyl acetate extract of Claviceps purpurea. Normal cellines of fibroblast (3T3L1) showed lesser % inhibition.
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MTT assay is a commonly believed in vitro method for screening the drugs having anticancer activity. In vitro anticancer activity against ethyl acetate extract of Claviceps purpurea on A549 cell line at different concentrations was evaluated. Standard vinblastine showed IC50 values are in MCF-7, 20.73 µM/mL, A549, 24.86 µM/mL, HeLa 21.23 µM/mL, and normal cell line Fibroblast (3T3L1) showed no IC50 values in all the cancer cell lines.
Cell cycle studies
Ethyl acetate extract in cell cycle studies, the results were found to be as followed in order to determine the cell cycle pattern of A549 cells, vinblastine and ethyl acetate extracts were treated to cells were analyzed in flow cytometric method. The cell cycle phase distribution was quantitated from 3 independent sets of measurements that showed relatively similar pattern to ethyl acetate extract which was different than standard Colchicine. The results were showed in figure 1-5 and table 2.
The treatment of cells at the concentration of 40µg/ml of ethyl acetate extracts showed on G0/G1 phase 77.11%, synthesis phase 11.56% and on G2 phase 7.98% of cells were arrested. The concentration of 80µg/ml 19.75% of cells were arrested on G2 phase of lung cancer (A549) cells when compared to standard Colchicine at 25 µM showed 44.22% of cells were arrested on G2 phase. According to (Angel et al., 2011) the cell cycle analysis revealed that the proportion of cells in the G2/M phase, this indicated that doxorubicin induced cell cycle arrest at the G2/M phase. The ethyl acetate extract of F 21- E (Aspergillus sp) induced an accumulation of cells showing prominent G2-M phase arrest. in the Sub-G0 phase (90.92 %).
The ability of anticancer agents to suppress the growth of cancer cells can also be associated with blocking cell-cycle progression. Based on the significant inhibition of A549 cell proliferation by ethyl acetate extract, it was postulated that the extract might modulate the cell-cycle progression of these cancer cells. Cell-cycle distribution analysis showed this extract caused a reduction in S phase population, which was associated with the accumulation of cells in the G0/G1 phase at a concentration of 40 µg/ml. A secondary metabolite of Epanorin (EP) exhibited a G1 cell cycle arrest which was similar in magnitude to that shown by TXM, a clinically used anti proliferative drug for estrogen positive breast cancer cells. Moreover, the effects on cell cycle progression were comparable to the reported for coumetarol on the MCF-7 Cells (Juan et al., 2019).
Apoptosis detection using PI Annexin V-FITC staining of A549 cells by flow cytometry
Apoptotic and necrotic cell populations in both control and treated cancer cells were studied by FACS using Annexin V-FITC and Propidium Iodide. The concentration of ethyl acetate extract 40 µg/ml and 80 µg/ml has induced necrosis in A549 cells with 27.54% and 35.54% cells respectively; and Standard Vinblastine showed 11.06% and 10.22% early and late apoptotic cells when compared to control cells with 56.11% also has shown necrosis of 22.61%. The results were showed in figure 6-10 and table 3.
Apoptosis is a programmed cell death characterized by cleavage of chromosomal DNA into oligonucleosomal fragments. Apoptosis is an active form of cell death that occurs in response to several agents, including chemotherapeutic drugs. Apoptosis was initially described by its morphological characteristics, including cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. Analysis of nuclear morphology is highly essential to examine the induction of cell death (Ramya et al., 2017).
DNA fragmentation studies on A549 cell line
DNA fragmentation is an important feature in cells undergoing apoptosis. To confirm that ethyl acetate of Claviceps purpurea makes cell death in A549 cells via apoptosis and not necrosis, DNA fragmentation patterns in treated cells were analyzed. The activation of endogenous endonucleases results in the cleavage of chromatin into inter-nucleosomal fragments of approximately 180–200 bp; it is believed that DNA fragmentation is carried out by Caspase-activated DNase (CAD), leading to the cleavage of nuclear DNA into multiple fragments with low molecular weights. A549 cells treated with two different concentrations of ethyl acetate extract for approximately 2hrs showed inter-nucleosomal DNA fragmentation in the form of a DNA ladder, typical of apoptotic DNA fragmentation (Fig 11). The exact DNA fragmentation and the absence of a DNA smear due to non-specific DNA degradation show the apoptosis-inducing ability of ethyl acetate extract. In this study, A549 cells were treated with Sample 2 (Ethyl acetate extract) and it was found that Sample has induced comparatively more DNA damage at 80 μg/ml when compared to Standard H2O2 with 200 µM concentrations.
The fragmentation of genomic DNA was confirmed by DNA laddering assay to support the apoptosis induction by the extract in the cancer cells. It was observed that the treated cells showed DNA laddering pattern and a single genomic DNA band was in untreated cells. The cells treated with control positive had also DNA laddering pattern.
In ethyl acetate extract of Claviceps purpurea showed significant result of DNA fragmentation studies in A549 cellines. The concentrations of 40µg/ml and 80µg/ml of treated cells were showed significant DNA fragmentation compare to control cells these treated cells showed better results. The standard H2O2 also showed more DNA damaged in cells and this activity may be contributed by the extract constituents. The fragmentation was not found in distinct, but increased DNA damage was observed, which provide evidence for apoptotic cell death.
Cancer chemotherapy drugs usually exert cytotoxic effects on malignant cells by inducing apoptosis. Major apoptotic events include cell cycle arrest and the formation of reactive oxygen species, leading to loss of mitochondrial membrane potential and DNA fragmentation. It is believed that caspase 3, a major downstream effector of apoptosis, mediates proteolytic cleavage of poly (ADP ribose) polymerase (PARP), leading to DNA fragmentation. These apoptotic events, accompanied by the potent growth inhibition of HeLa cells, were detected when the cells were treated with partially purified VCR from T. radicus—CrP20 (Padmini et al., 2015).
Dual staining was examined under a fluorescent microscope. Normal cells are seen with circular nucleus uniformly distributed in the center of the cell which is seen in the control figure 12.
In treated sample 40 μg/ml show in the early stage apoptotic cells, where cell’s nucleus is showing yellow-green fluorescence by Acridine orange (AO) staining and concentrated into a crescent or granular that located in one side of cells. Staining was localized asymmetrically within the cells and membrane blebbing was seen in figure 13.
In treated sample 80 μg/ml show in the nucleus of cells were orange fluorescence by EtBr staining and gathered in concentration and located in bias, this is late apoptotic phase. Cells that have taken up complete EtBr are the necrotic cells as in Figure 14. In standard Vinblastine at 25 µM/ml showed on A549 cellines in figure 15.
Ethyl acetate extract of Claviceps purpurea showed at 40 µg/ml concentration yellow-green fluorescence by Acridine orange (AO) staining and concentrated into a crescent or granular that located in one side of cells. Staining was localized asymmetrically within the cells. At 80 µg/ml concentration orange fluorescence by EtBr staining and gathered in concentration and located in bias. This is late apoptotic phase in A549 cell lines. Membrane blebbing will be found. It indicates ethyl acetate extract of Claviceps purpurea is very active against cancer cell lines.
According to Tayyaba et al., 2018 state that the results of acridine orange (AO) and ethidium bromide (EB) staining of cells treated with AHC. The results of acridine orange (AO) and ethidium bromide (EB) staining of cells treated with AHC. AO is a cell permeable fluorescent dye and stains nuclear DNA in both live and dead cells, whereas EB is a fluorescent dye that only stains nuclear DNA in cells that have lost their membrane integrity. We observed that after AO/EB staining viable cells were uniformly stained green, early apoptotic cells were stained greenish yellow or displayed green yellow fragments, late apoptotic cells were stained orange or displayed orange fragments, and necrotic cells showed orange to red fluorescing nuclei with no indication of chromatin fragmentation.