Table of Contents    
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 2  |  Page : 131-138  

Phytochemical screening and evaluation of cytotoxic activity of Calotropis gigantea leaf extract on MCF7, HeLa, and A549 cancer cell lines


1 Department of Medical Research, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu; Department of Genomics, Genei Laboratories Private Limited, Bengaluru, Karnataka, India
2 Department of Research and Development, Clovergen Life Sciences Private Limited and Scientific Bio Minds, Bengaluru, Karnataka, India
3 Department of Genomics, Genei Laboratories Private Limited, Bengaluru, Karnataka, India
4 Department of Medical Research, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, Kattankulathur, Tamil Nadu, India

Date of Web Publication18-Jul-2019

Correspondence Address:
Venkataraman Prabhu
Department of Medical Research, SRM Medical College Hospital and Research Centre, SRM Institute of Science and Technology, Kattankulathur - 603 203, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_215_18

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   Abstract 


Background: Cancer is mostly managed by surgical removal, chemotherapy, and radiotherapy. However, there are side effects associated with these methods. Alternatively, herbal medicines are becoming popular for cancer treatment. Calotropis gigantea is a widely used plant in the traditional medical system. However, there are no reports on its potential in cancer management. Therefore, we aimed to examine the phytochemical composition and cytotoxic activity of C. gigantea methanolic leaf extract against three different cancer cell lines: HeLa (cervical), MCF7 (breast), and A549 (lung). Materials and Methods: The methanolic extract of C. gigantea leaf was used to determine the presence of phytoconstituents using standard methods such as thin-layer chromatography, high-performance liquid chromatography, and liquid chromatography–mass spectrometry. HeLa, MCF7, and A549 cell lines were treated with C. gigantea methanolic leaf extract at different concentrations (0, 100, 200, 300, and 400 μg/mL). Camptothecin and cisplatin were used as reference drugs for growth inhibition studies. Results: The phytoconstituents of methanolic leaf extract of C. gigantea included alkaloids, steroids, terpenoids, flavonoids, tannins, and phenols. The extract exhibited cytotoxicity against HeLa (IC50= 117.92 μg/mL), MCF7 (IC50= 43.65 μg/mL), and A549 (IC50= 27.32 μg/mL) cancer cell lines in vitro. Conclusion: Our results indicated that C. gigantea exhibited cytotoxicity against cervical, breast, and lung cancer cell lines in vitro, and thus, the crude extract can be a potential candidate for cancer treatment.

Keywords: Calotropis gigantea, cancer, herbal medicines, phytochemicals


How to cite this article:
Damodaran B, Nagaraja P, Jain V, Manuja Viraj Wimalasiri M P, Sankolli G M, Kumar G V, Prabhu V. Phytochemical screening and evaluation of cytotoxic activity of Calotropis gigantea leaf extract on MCF7, HeLa, and A549 cancer cell lines. J Nat Sc Biol Med 2019;10:131-8

How to cite this URL:
Damodaran B, Nagaraja P, Jain V, Manuja Viraj Wimalasiri M P, Sankolli G M, Kumar G V, Prabhu V. Phytochemical screening and evaluation of cytotoxic activity of Calotropis gigantea leaf extract on MCF7, HeLa, and A549 cancer cell lines. J Nat Sc Biol Med [serial online] 2019 [cited 2019 Aug 20];10:131-8. Available from: http://www.jnsbm.org/text.asp?2019/10/2/131/262953




   Introduction Top


Cancer is the growth of tissues characterized by the uncontrolled division of cells and they severely affect the human population. According to the World Health Organization, cancer is the second leading cause of death globally and was responsible for an estimated 9.6 million deaths in 2018.[1] Globally, about 1 in 6 deaths is due to cancer. In India, it is estimated that around 2.25 million people are suffering from different types of cancer according to the National Institute of Cancer Prevention and Research.[2] The cost associated with cancer treatment is expected to rise to 7%–10% per annum by the year 2020, when global costs will go beyond $150 billion.[3],[4] Different plants have long been used for the management of numerous diseases. Most of the herbal medicines have been used to treat many diseases in most developing countries.[5],[6] Understanding the core concepts of plant usage in traditional medicine as therapeutic agents can help in identifying novel molecules for treating many diseases, especially cancer.

The plant, Calotropis gigantea, is used in traditional medical systems such as Ayurveda, homeopathy, and traditional Chinese medicine. This species is commonly known as a crown flower or giant milkweed. It is a large shrub found abundantly in Asia and Africa.[7],[8] The plant is used to treat conditions such as fever, cold, cough, diarrhea, indigestion, asthma, leprosy, and leukoderma.[9],[10] The extract of different parts of C. gigantea has been reported to have many promising primary and secondary metabolites and can be separated or combined for the development of effective drugs against various types of cancer.[11]

Phytochemicals are biologically important molecules that play a critical role in the plant's defense system. Many selective phytochemicals including primary and secondary metabolites have been developed as drug molecules. Carbohydrates, chlorophyll, proteins, and lipids form the primary metabolites, while the secondary metabolites include alkaloids, tannins, saponins, phenols, flavonoids, and terpenoids. The secondary metabolites have been described as potent anticancerous agents and as important cancer chemopreventive agents.[12] Polyphenols, flavonoids, and steroids are some of the phytochemicals that have been extensively studied as anticancerous agents.[6],[13] These secondary metabolites have also been described as pharmacological agents or noncytotoxic nutrients that boost the physiological mechanisms of an organism.[14] However, understanding the cytotoxic nature of the metabolites is important to evaluate their anticancer activity.

The biological activity, including anticancer activity, of metabolites can be evaluated using bioassays and in vitro techniques. Currently, various cytotoxicity assays are based on the quantification of cell proliferation, assessing cell membrane integrity, or measuring protein content in cells, which include trypan blue dye exclusion assay, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, and sulforhodamine B assay.[15] These assays help in assessing the cytotoxicity of the metabolites in cells. Although the plant kingdom has been investigated for a number of compounds, there are ongoing efforts to identify novel anticancer molecules.

The aim of the study was to analyze the phytochemical properties of C. gigantea and to assess the cytotoxic activity of the leaf extracts using in vitro assays against HeLa, MCF7, and A549 cancer cell lines.


   Materials and Methods Top


Cell culture

HeLa (cervical), MCF7 (breast), and A549 (lung) cell lines were purchased from the National Centre for Cell Science, Pune, and cultured in Dulbecco's modified Eagle's high-glucose medium (DMEM; Cat No. AL111, HiMedia, Mumbai, India) supplemented with 10% (v/v) fetal bovine serum. Camptothecin and cisplatin were used as reference drugs for growth inhibition studies. All commonly used drugs and chemicals (analytical grade) were purchased from HiMedia, Mumbai, India, and Sigma Chemical Co., USA.

Identification and collection of plant materials

Fresh and healthy leaves of C. gigantea were collected from Yelahanka, Bengaluru, Karnataka, India. The plant was taxonomically identified and confirmed (specimen Ref No: 4588) by a trained botanist at University of Agricultural Sciences, Bengaluru, Karnataka, India. The plant leaves were cleaned to remove extraneous matter, and necrotic parts were removed by rinsing in fresh water and running distilled water.

Preparation of methanol extracts

Fresh green leaves (1 kg) of C. gigantea were shade dried and powdered by pounding and grinding. Powdered leaf samples were sieved, weighed, and stored in an airtight container. The powder (3 g) was measured and mixed with 30 mL of methanol [16] in a conical flask. For extraction, the conical flask was plugged with a cotton plug and was placed in a shaker at 28°C for 24 h at 150 rpm. The mixture was filtered through muslin cloth and Whatman (No. 1) filter paper to obtain the extract. The green sticky mass of 200 g extract was labeled and preserved in the refrigerator at 4°C until use.[17] Suspensions of the extract were freshly prepared for experimental use.

Experimental design

The three different cancer cell lines were cultured and treated with the C. gigantea leaf methanol extract. The cytotoxic activity was measured using MTT assay. The percentage of growth inhibition was calculated using the formula mentioned under cytotoxicity assay section. The concentration of test drug needed to inhibit cell growth by 50% (IC50 value) was generated from the dose-response curve for each cell line.

Phytochemical screening

C. gigantea leaf methanol extract was subjected to successive standard phytochemical screening following the method of Harborne (1983) to identify alkaloids, steroids, terpenoids, flavonoids, tannins, and phenols.[18] All phytochemicals were carefully screened and identified by characteristic color changes following standard procedures.

Thin-layer chromatography

The crude extract was subjected to thin-layer chromatography (TLC) to detect and separate the chemical constituents of the crude extract and its fractions (stationary phase: silica gel F254 and mobile phase: methanol: water [99:1 v/v]). The spots visible both under visible light and ultraviolet light were recorded. The respective retention factor (RF) values for all the spots were calculated.

Column chromatography

The crude methanolic extract was subjected to column chromatography to isolate the active compound. A clean, dried column was aligned in a vertical position. A beaker was placed under the column outlet. The column was rinsed with methanol, and a loose plug of cotton, which had been washed with methanol, was tamped down at the bottom of the column. The stationary phase was prepared by filling the column with silica gel slurry, which was prepared using methanol, till the desired height was attained. One milliliter of the extract was loaded into the column packed with silica gel. Methanol (mobile phase) was poured continuously from the top of the column using a funnel. The bottom outlet of the column was opened, and different fractions of the elute were collected in separate test tubes.[19]

High-performance liquid chromatography

The crude methanolic fractions of the C. gigantea leaf samples were separated using an analytical high-performance liquid chromatography (HPLC) system (Shimadzu) fitted with 200 mm × 4.6 mm C18 column. Methanol (HPLC grade, 0.2 μm filtered) was used to prepare the mobile phase (methanol: water – 75:25 v/v). The compounds were separated at a flow rate of 1.0 mL/min and column temperature of 30°C with an injection volume of 40 μL. The detection was carried out at 254 nm, 284 nm, and 234 nm.[20]

Liquid chromatography–mass spectrometry

The samples were subjected to HPLC (Perkin Elmer 200 HPLC system) and mass spectrometry (AB Sciex API 3000 liquid chromatography–mass spectrometry (LCMS) system). The mobile phase A constituted methanol while mobile phase B constituted bidistilled water with 0.1% (v/v) formic acid. The mobile phase, which was previously degassed with helium, was pumped at a flow rate of 0.3 mL/min and an injection volume of 10 μL. The electrospray ionization (ESI) mass spectrometer was operated in the positive ion mode. The electrospray capillary potential was set to 50 V while the shield was at 225 V. Nitrogen at 49 mTorr was used as a drying gas for solvent evaporation. The atmospheric pressure ionization housing and drying gas temperatures were kept at 50°C and 380°C, respectively. The scan time was 1 s and the detector multiplier voltage was set to 1500 V with an isolation width of m/z 1.2 for quadrupole 1 and m/z 2.0 for quadrupole 3. The ESI mass spectra were acquired by scanning at a mass range of 50–700.[21]

Cytotoxicity assay

The crude methanolic extract was air-dried in a petri dish for 2 days at 28°C under sterile conditions. The extract was then scraped into a sterile vial. Different concentrations of the leaf extract were used for performing MTT assay. HeLa, MCF7, and A549 cancer cell lines were cultured and maintained until they reached 70% confluency in T25 flask at 37°C in a 5% CO2 incubator. Further, 200 μL cell suspension was seeded in a 96-well plate at the required cell density (20,000 cells per well), without the test agent, and the cells were allowed to grow for about 24 h. The methanolic extract was dissolved in DMEM at the required concentrations (25, 50, 100, 200, and 400 μg/mL) and incubated for 24 h at 37°C and 5% CO2.[22] Camptothecin and cisplatin at a concentration of 25 μM were used as a positive control for the study. After the incubation period, the spent medium was removed, and 100 μL MTT reagent (Cat No: 4060, HiMedia Mumbai, India) was added to the cells and incubated for 3 h at 37°C. The resulting formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO; Cat No. 1309, Sigma, USA) and its absorbance was measured using microplate plate reader (ELx800, BioTek) at 570 nm. The IC50 value was calculated using the linear regression equation (y = mx + c) obtained from the cell viability graph.

The viability of the cells was determined by the following formula:

Percentage viability = (OD570 nm of test compound treated cells/OD570 nm of untreated cells) ×100.

Based on the MTT assay results, the most effective cell line A549 was further taken for apoptotic and cell cycle study for analysis of cytotoxicity.

Study of Calotropis gigantea leaf methanolic extract on apoptosis of A549 cell line

A549 cells were cultured in a 6-well plate at a density of 2 × 105 cells/mL and incubated at 37°C for 24 h. The cells were then treated with 27.32 μg/mL leaf extract (cytotoxic IC50) and 25 μM cisplatin in 2 mL DMEM and incubated for 24 h. The spent medium was removed, and the cells were washed with 2 mL of 1X phosphate-buffered saline (PBS). Cells in each well were resuspended in 500 μL trypsin and incubated at 37°C for 3–4 min. Two milliliters of culture medium was added to the cells, and the cell suspension was transferred into 12 × 75 mm tubes. The cells were centrifuged for 5 min at 300 g at 25°C (REMI R-8C, REMI, India). The supernatant was discarded and the cells were washed using 1 mL PBS. Cells treated with cisplatin served as a positive control. To the cells, 5 μL FITC Annexin V and 5 μL propidium iodide were added along with 100 μL of 1X Annexin V Binding Buffer. The cells were incubated for 15–20 min, and 300 μL 1X Annexin V Binding Buffer was added. The cells were analyzed immediately using a flow cytometer (BD FACSCalibur, BD Biosciences).

Study of Calotropis gigantea leaf methanolic extract on the cell cycle of A549 cell line

A549 cells were cultured in a 6-well plate at a density of 2 × 105 cells/2 mL and incubated at 37°C for 24 h. The cells were treated with 27.32 μg/mL leaf extract (cytotoxic IC50) and 25 μM cisplatin in 2 mL DMEM and incubated for 24 h. The spent medium was removed and the cells were washed with 2 mL 1X PBS. Cells in each well were resuspended in 500 μL trypsin and incubated at 37°C for 3–4 min. Two milliliters of culture medium was added to the cells that were transferred directly into 12 mm × 75 mm tubes. The cells were centrifuged for 5 min at 300 g at 25°C (REMI R-8C, REMI, India). The pelleted cells were washed with 1 mL of PBS. The cells were then fixed and permeabilized using 70% of prechilled ethanol for 30 min in −20°C deep freezer. The cells were centrifuged to remove ethanol and washed with 1X Dulbecco's PBS. The cells were stained using 400 μL propidium-RNAse solution (BD Biosciences, USA) for 20–30 min in dark at room temperature (28°C–30°C). The cells were analyzed immediately using a flow cytometer (BD FACSCalibur, BD Biosciences).

Statistical analysis

Data were expressed as mean ± standard error of mean. Statistical significance was evaluated by one-way ANOVA using SPSS version 2 (IBM Corp., Armonk, NY, USA). In all cancer cell lines, P < 0.05 was considered as statistically significant, and the IC50 value was determined using linear regression equation, i.e., y = mx + c (y = 50, m, and c values were derived from the viability graph).


   Results Top


We screened the methanolic leaf extract of C. gigantea for its phytochemical constituents following standard protocols. We observed a higher abundance of alkaloids, terpenoids, and flavonoids as compared to the abundance of steroids and phenols in the extract. Trace amounts of glycosides were also observed [Table 1]. Further, the crude extract was subjected to TLC. We observed seven different bands of varying colors including light yellow, light green, and dark green. The RF values calculated for the spots are shown in [Table 2]. The samples were also subjected to column chromatography, and the resultant fractions were used for HPLC analysis. The crude extracts were further purified using analytical HPLC. Alkaloid components were detected at 254 nm, steroids and flavonoids at 234 nm, and phenols, tannins, and terpenoids at 284 nm [Figure 1]a, [Figure 1]b, [Figure 1]c and [Table 3]. The LCMS spectral results and its comparison with the library successfully identified several chemical compounds [Figure 2]a and [Figure 2]b including glycosides, alkaloids, and terpenoids. We identified 17 compounds based on ESI-positive scan along with their respective molecular weights using alkali metal cationization [Table 3]. Major compounds such as holarrhetine (372.60), calotropogenin (404.50), beta-sitosterol (414.42), taraxasterol (426.73), sapogenins (470.69), calactin (532.63), calotoxin (548.63), cyanidin (595.53), uscharin (587.74), and quercetin-3-rutinoside (610.52) along with their metabolites were reported previously in the plant extract.[23],[24]
Table 1: Phytochemical analysis of methanolic leaf extract of Calotropis gigantea

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Table 2: Retention factor values of the spots observed in thin-layer chromatography

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Figure 1: (a-c) The pattern of ultraviolet light absorption in high-performance liquid chromatography analysis of the methanolic extract of Calotropis gigantea

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Table 3: High-performance liquid chromatography and liquid chromatography–mass spectrometry analysis of chemical constituents in the methanolic leaf extract of Calotropis gigantea

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Figure 2: (a and b) Liquid chromatography–mass spectrometry analysis for the electrospray ionization positive metabolites of Calotropis gigantea extract

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The methanolic extract of C. gigantea exhibited cytotoxic effect against all three cancer cell lines. HeLa, A549, and MCF7 cell lines exhibited varying levels of viability upon treatment with different concentrations of the extract. IC50 value (117.92 μg/mL) of the extract was found to be higher in the HeLa cell lines [Table 4] and [Figure 3], [Figure 4], [Figure 5], [Figure 6] compared to the IC50 value in A549 (27.32 μg/mL) and MCF7 (43.65 μg/mL) cells. The positive control, cisplatin, exhibited the highest cytotoxicity against HeLa (IC50= 6.3 × 10−4 μg/mL) cell line, followed by MCF7 (IC50= 2 × 10−1 μg/mL) and A549 (IC50= 8 × 10−1 μg/mL). We used A549 cell lines for further analysis because the extract exhibited the highest cytotoxic effect in this cell line. We evaluated the potential of the extract to induce apoptosis in A549 cells by flow cytometry analysis. We observed that the extract predominantly induced late apoptotic cell death (84.92%) in the A549 cell line. In addition, the extract induced early cell death in a small proportion (2.37%) of cells. A similar trend was observed with the positive control (25 μM cisplatin), which predominantly induced late apoptotic cell death (92.22%) and induced early apoptotic cell death in a small proportion of cells (3.1%) [Table 5] and [Figure 7].
Table 4: Half maximal inhibitory concentration values of MCF7, A549, and HeLa cells treated with methanolic leaf extract of Calotropis gigantea

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Figure 3: Cytotoxic effect of Calotropis gigantea methanolic leaf extract against MCF7 cancer cell line

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Figure 4: Cytotoxic effect of Calotropis gigantea methanolic leaf extract against A549 cancer cell line

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Figure 5: Cytotoxic effect of Calotropis gigantea methanolic leaf extract against HeLa cancer cell line

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Figure 6: Comparative analysis of the IC50 value of the test compound against the MCF7, A549, and HeLa cancer cell lines. Comparative cytotoxicity of Calotropis gigantea methanolic leaf extract. The IC50 values obtained after 24-h drug treatment in MCF7, A549, and HeLa cell lines are shown. The extract was highly cytotoxic to A549 cell line

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Table 5: Apoptosis study by Annexin V-PI expression of the test compound 1 (methanolic leaf extract [methanolic extract]) against A549 cell line

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Figure 7: Annexin V-PI expression study of the test compound 1 (Methanolic leaf extract) against A549 cell line: Annexin V-PI expression study of test compounds on A549 cells using BD FACS Calibur, Cell Quest Pro Software (Version 6.0). Quadrants showing the expression of Annexin V-PI in untreated cells (a), 25 μM cisplatin treated cells (b) and Calotropis gigantea methanolic leaf extract treated cells (c). In each figure, the upper left quadrant indicates the percentage (%) of necrotic cells and the upper right quadrant indicates the percentage (%) of late apoptotic cells. Bottom left quadrant indicates the percentage (%) of viable cells and bottom right indicates the percentage (%) of early apoptotic cells

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We also studied the effect of the extract on the cell cycle of A549 cells. We observed that the extract induced cell cycle arrest at S phase in A549 cells when compared to cisplatin [Table 6] and [Figure 8].
Table 6: Effect of Calotropis gigantea methanolic leaf extract against A549 cell line

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Figure 8: Cell cycle study of the test compound (methanolic leaf extract) against A549 cell line: Cell cycle analysis of test compound against A549 cells using BD FACSCalibur. Propidium iodide histogram of the gated cell singlet distinguishing the cells at the sub-G0/G1, G0/G1, S, and G2/M cycle phases. a) Untreated cells, b) Cell cycle analysis with standard drug, c) Cell cycle analysis with Calotropis gigantea methanolic leaf extract

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   Discussion Top


Plants have a rich source of phytochemicals that can be potential therapeutics for treating various diseases. Ayurveda is a traditional medical system that also involves the use of plant-based therapeutics. In India, ayurvedic drugs are used in the treatment of various diseases, including cancer. The therapeutic effect of the plants is mainly attributed to their secondary metabolites. In this study, we screened the methanolic leaf extract of C. gigantea for their phytochemical constituent. We observed an increased abundance of alkaloids, terpenoids, and flavonoids in the extract. These secondary metabolites are previously reported to exhibit antiproliferative and antimetastatic effects [25] through activation of the apoptotic pathway in cancer.[26] Hence, we also focused on evaluating the cytotoxic, antiproliferative, and proapoptotic effects of C. gigantea leaf extracts on cancer cell lines. We demonstrated that C. gigantea leaf extracts are cytotoxic against HeLa, MCF7, and A549 cell lines. Among these cell lines, the extract exhibited the highest cytotoxicity against the lung cancer cell line, A549 (IC50= 27.32 μg/mL), followed by MCF7 (IC50= 43.65 μg/mL) and HeLa (IC50= 117.92 μg/mL) cell lines. Hence, we used A549 cell line to further analyze the cytotoxic mechanisms of the extract.

Cell cycle checkpoints play an important role in preventing the cells from entering the next stage of cell cycle in case of DNA damage. In cancer cells, the cell cycle checkpoints are impaired and thus proceed with the cell cycle despite DNA damage which leads to uncontrolled proliferation.

Alkaloids, terpenoids, and flavonoids are known to exert its antiproliferative effect by inducing cell cycle arrest.[27],[28],[29] We observed that the C. gigantea leaf extract, which is rich in these secondary metabolites, induced cell cycle arrest at S phase, similar to cisplatin, in the A549 cell line.

In addition, plant secondary metabolites are also reported to activate apoptotic pathways.[30] Hence, we hypothesized that the underlying cytotoxic mechanism of C. gigantea also involves the activation of apoptotic pathway. Indeed, we observed that the C. gigantea leaf extracts can induce apoptosis in the A549 cell line. Specifically, the extract predominantly induced late apoptosis, similar to cisplatin, in the A549 cell line.

The cytotoxic effects of the methanolic C. gigantea leaf extracts may be due to the activation of apoptotic pathway in lung cancer cell lines. However, it is unclear why the extract exhibited lower cytotoxicity in the breast cancer and cervical cancer cell line. Further studies are required to unravel the specificity of the extract on lung and other cancer cell lines.


   Conclusion Top


This study was screened for the phytochemical constituents of the methanolic C. gigantea leaf extract. The presence of active compounds that belong to flavonoids, alkaloids, and terpenoids indicates the medicinal importance of the plant. In vitro cytotoxic assay supports the cytotoxic potential of the compounds present in the leaf extracts of C. gigantea. The extract induced apoptosis in lung cancer cell line. Further screening of the active compounds using in silico methods will help to understand the pharmacological activity of the compounds against cancer cell lines.

Acknowledgments

Dr. Chandrasekhar, Genei Laboratories, and Dr. C. N. Prashantha for in silico studies are gratefully acknowledged. Dr. Akash Navilebasappa, Acquity Labs, is also acknowledged for supporting in LCMS experiment and analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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