Phytochemicals from Corchorus olitorius methanolic extract induce apoptotic cell death via activation of caspase-3, anti-Bcl-2 activity, and DNA degradation in breast and lung cancer cell lines

2.1 Plant material and extraction

The whole plant of C. olitorius was obtained in March 2021 from an authentic supplier in Najran local market, Saudi Arabia, and identified by an expert pharmacognosist, Dr Mohammed A. A. Orabi, Associate Professor of Pharmacognosy, Pharmacy College, Najran University, Najran, Saudi Arabia, and a herbarium specimen (CorchL-03–2021) was deposited in the Department of Pharmacognosy, College of Pharmacy, Najran University.

In the current study, the Soxhlet extraction method was used in the sequential extraction with the solvents hexane, ethyl acetate, methanol, and distilled water, based on their increasing order of polarity. Around 100 g dry plant sample of C. olitorius was extracted with solvents. Approximately 1000 ml of the extraction solvent was used. The extracted plant material was dried before being extracted with the successive solvents.

2.2 Phytochemical analysis

Phytochemical screening was performed for detection of presence and absence of tannins, flavonoids, steroids, terpenoids, alkaloids, glycosides, phenols, anthraquinones, based on previously prescribed procedures (Roghini and Vijayalakshmi, 2018).

2.3 Estimation of total phenolic and flavonoid content

Using a spectrophotometric method, the plant extract's total phenolic compounds was determined (Singleton et al., 1999). Gallic acid (GA) calibration curves were created in the 20–100 μg/ml range. Finally, the concentrations of phenolics were converted to Gallic acid equivalents:μg GAE/g of dry weight (dw) of the extract. The total phenolic content was calculated from the standard calibration curve (Y = 0.001X + 0.113).

Further, aluminum chloride colorimetric assay was used to determine total flavonoid content (Zhishen et al. 1999). 20–100 µg/ml standard quercetin was developed. A 10 ml volumetric flask containing 4 ml double distilled water was added to 1 ml of each quercetin concentration in methanol. At time zero, 0.3 ml of 5 % sodium nitrite, 0.3 ml of 10 % AlCl3, and 2 ml of 1 M sodium hydroxide were added. Immediately, 2.4 ml double-distilled water was added to the mixture and thoroughly stirred. The pink color mixture's absorbance was measured at 510 nm against a quercetin-free blank. The calibration curve was plotted using average quercetin absorbance values. The concentrations of flavonoids were converted to quercetin equivalents: μg QE/g dw of the extract (Y = 0.265x − 0.152).

2.4 Gas chromatography-mass spectrometry (GC–MS) profiling

GC/MS analysis was performed for the methanol extract of C. olitorius using GCMS-QP2010S model. The extract was administered in the sample injector of GC–MS apparatus in a 1 µl aliquot. The column temperature was initially set at 80 °C, while the injector was set at 260 °C, and the flow temperature was set to increase at a rate of 10 °C/min throughout the process. In case of GC, ion source temperature and interface temperature was 200 °C and 280 °C respectively. Solvent cut time was set to 6.50 min with relative detector scan mode. In case of MS, start time and end time was 7 min and 50 min, respectively, under scan mode with 0.50 sec time. Scan speed was 1000 and start m/z and end m/z was 50 and 500 respectivley. The mass spectra of the components were compared to those in the NIST mass spectral library to identify them (Davies, 1990).

2.5 Liquid chromatography-mass spectrometry (LC-MS) profiling

LC-MS-8040 (Shimadzu) model was used to analyze and identify different chemical constituents in the methanol extract of C. olitorius. The HPLC was coupled with mass spectra. A C18 column was used with a mobile phase methanol/water (80:20, v/v). The mobile phase constant flow rate was maintained at a rate 0.2 ml/min. The Electron Spray Ionization probe was used as detector mode, a 3 µl of the sample was used for the analysis (Hanafi et al., 2018). In case of MS the ESI (electron spray ionization) probe was used with M + 1 and M−1 ionization variation.

2.6 In-vitro cytotoxicity of C. Olitorius extracts

Cytotoxicity assay was performed by following the standard procedure of MTT. In the current study, different solvent extracts of C. olitorius were taken in various concentrations (50, 100, 150, 200 and 250 µg/ml) for treating on the non-cancerous cell line, lung cancer (A549) and breast cancer (MCF-7) cell lines. IC₅₀ value was determined with the basis of cell viability percentage from the flowing equation (van Meerloo et al., 2011). $$\mathit{Inhibition}percentage=\left(O.D.oftestsample÷O.D.ofcontrol\right)\times 100$$

2.7 Flow cytometry based apoptosis study

In the present study, early and late apoptosis detection and measurement were performed by using IC₅₀ concentration of methanol extract of C. olitorius against MCF-7 and A549 cells with 24 hrs duration from the instructions provided in the performing kit. After incubation (24 h), analysis was done using FlowJoX 10.0.7 software (Crowley et al., 2016).

2.8 Caspase-3 activity

Flow cytometry was used to assess the activation of caspase-3 in MCF-7 and A549 cells by C. olitorius methanolic extract (Moraes et al., 2013). After treatment with test sample at its IC₅₀ concentrations. Fluorescence was measured after incubation for 24 h (Acquisition: 7. Cytomics FC500 Flow cytometer, Beckman Coulter, USA).

2.9 Anti Bcl-2 activity

Flow cytometry was used to quantify the level of Bcl-2 protein in treated MCF-7 and A549 cells by C. olitorius methanolic extract (Moraes et al., 2013). After treatment with test sample at its IC₅₀ concentrations, cell preparations was analyzed within 30 min by flow cytometry using FlowJo X 10.0.7 software.

2.10 DNA fragmentation analysis

The apoptotic DNA degradation induced by methanol extract of C. olitorius was performed by the TUNEL assay kit (The APO-DIRECT™ Kit) (Kyrylkova et al., 2012). Finally, using flow cytometry, the TUNEL-positive cells percentage was calculated using Flowzo 10.1 Acquisition: 7 Cytomics FC500 Flow cytometr, (Beckman Coulter, USA).

2.11 Statistical analysis

The data were analyzed using a one-way ANOVA followed by Dunnett's test using GraphPad Prism Version 5. p < 0.05 was chosen as the level of statistical significance. The results are presented as the mean standard deviation for three separate tests (n = 3).

3.1 Phytochemical analysis and quantification

The phytochemical analysis revealed the presence of flavonoids, terpenoids, phenols and saponins in the methanolic and aqueous extracts (Table.1). The quantification results showed that the methanol extract exhibited the higher phenolic (699 µg GAE/g of dw) and flavonoid (1361.50 µg QE/g dw) content than the aqueous extract which showed lower phenolic (421.6 µg GAE/g of dw) and flavonoid (1147.54 µg QE/g dw) content.

3.2 GC–MS analysis

Methanol extract of C. olitorius have shown the presence of 1-Tridecene, 1-Tetradecanol, Tetradecane methyl arachate, (Cis)-2-Nonadecene, Cyclopropane octanoic acid, Linolenic acid methyl ester, Phytol acetate, dibromo-stigmasterol acetate, methyl commate-D (Table 2 and Fig. 1).

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Fig. 1

GC–MS profiling of methanol extract of C. olitorius.

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3.3 LC-MS analysis

The results of the LC-MS analysis of methanol extract revealed the presence of 5-(3-Furan-2-yl-acryloyl)-2,2-dimethyl-4,6-dioxo-cyclohexanecarboxylic acid methyl ester and 1-(8-Quinolinyl)-beta-carboline as major compounds (Table 3 and Fig. 2, S14). These compounds have the chemical nature of esters and alkaloids.

Table 3 LC-MS based profiling of methanol extract of C. olitorius.

Sl.no	Structure	Compound name	Retention time	Nature	Uses
1		5-(3-Furan-2-yl-acryloyl)-2,2-dimethyl-4,6-dioxo-cyclohexanecarboxylic acid methyl ester	9.538	Ester	Industrial application
2		1-(8-Quinolinyl) -beta-carboline	12.136	Alkaloid	Induce brain function and antioxidant

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Fig. 2

LC-MS analysis of methanol extract of C. olitorius.

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3.4 In-vitro cytotoxicity

In this assay, the different concentrations (50, 100, 150, 200 and 250 µg/ml) of the hexane, ethyl acetate, methanol and aqueous extracts were used to treat L929 cell line to check the percentage of viability. The results revealed that the ethyl acetate extract had shown a high level of toxicity among all tested extracts. The results are shown in Table 4. The morphological effects of MTT assay for each solvent extract are provided as Supplementary Files (Figures S1–S4).

3.5 In-vitro cytotoxicity against MCF-7 and A549 cell lines

At the initial concentration of hexane, ethyl acetate, methanol, and aqueous extracts, the cell viability in lung cancer was observed to be 80.91 ± 0.008, 77.19 ± 0.003, 45.97 ± 0.003 and 59.06 ± 0.004 % (Table 5). In the case of breast cancer, it was found to be 71.86 ± 0.0145, 51.64 ± 0.0075, 45.70 ± 0.001 and 68.51 ± 0.001 % (Table 6). Further, the IC₅₀ value of test samples was calculated from the standard calibration curve. In the case of hexane, ethyl acetate, methanol and aqueous extracts for breast cancer, it was observed to be 182.47, 55.18, 20.0, and 126.58 %, respectively. In the case of lung cancer, it was found to be 245.15, 227.46, 12.45, and 79.47 %, respectively. The IC₅₀ details have been shown in Table 7.

The morphological study also correlates with MTT results. In both treated MCF-7 and A549 cell lines, the test samples have shown changes in the cellular morphology compared to the untreated cells. In the untreated cells, the morphology was clear, and they were in more numbers without any intracellular spaces. Whereas, in test samples treated cells, as the concentration was increased, the cell number gradually decreased, and there was an observation of intracellular spaces between the cells (Supplementary file: Figures S5–S12).

3.6 Detection of early and late apoptosis

The results revealed that there was no sign of apoptosis in the case of untreated cells, whereas, in the case of the test treated MCF-7 cells, around 4.18 % and 5.72 % of cells showed the positive result for the early and late apoptosis, respectively (Figure S15). Similarly, in the case of standard drug cisplatin-treated cells, it was observed to be 1.96 % and 8.65 %. The study in lung cancer A549 cells revealed that the methanol extract was inducing apoptosis similar to the standard drug cisplatin. In the case of methanol extract-treated cells, early apoptosis-positive cells were 4.55 %, and late apoptosis cells were 11.30 %, in total, 15.85 % of cells were positive for apoptosis (Figure S16).

3.7 Caspase-3 assay

The results revealed that in the untreated group, around 6.24 % of cells showed positive for caspase-3 in MCF-7, and in lung cancer it was 4.94 %, it might be due to the natural properties of cells to have programmed cell death, and in cancer cells, this mechanism of programmed cell death can be seen but at a lower rate. Compared to the untreated, in the test sample and standard drug cisplatin, this percentage of caspase-3 positive cells increased. In the case of methanol extract treated breast cancer MCF-7, around 25.60 % of cells, and in lung cancer A549, 11.30 % of cells were positive for caspase-3 presence. In the case of standard drug cisplatin for breast cancer MCF-7, it was found to be 51.10 %, and it was 16.20 % for lung cancer (Figures S17 and S18).

3.8 Anti Bcl-2 inhibition assay

The results revealed that in the case of breast cancer MCF-7 untreated cells, a minor degree of apoptosis was observed, which showed the high-level Bcl-2 cells (89.40 %) and low-level Bcl-2 cells (6.28 %); and 92.20 % high level Bcl-2 positive cells and 7.12 % low level Bcl-2 positive cells were observed in lung cancer A549. Compared to the untreated in both test sample and standard drug cisplatin-treated group of cells, there was a change in the percentage of cells for low Bcl-2 and high Bcl-2. In the test sample treated for breast cancer MCF-7, low Bcl-2 positive cells were 14.70 %, and high BCL-2 positive cells were 76.00 %. Similarly, for lung cancer A549, low Bcl-2 positive cells were 12.40 %, and high Bcl-2 positive cells were 86.20 %. But in the case of standard drug cisplatin treated group of cells, significant changes in the Bcl-2 level i.e. for breast cancer MCF-7, low Bcl-2 was seen in 36.90 % of cells and high-level Bcl-2 seen in 52.20 % of cells. Similarly, for lung cancer A549, low-level Bcl-2 was seen in 35.60 % of cells, and 59.90 % of cells showed positive for the high level of Bcl-2 (Figures S19 and S20).

3.9 DNA damage study by TUNEL assay

The results revealed that in the untreated group of cells of breast cancer MCF-7 and lung cancer A549, around 5.99 % and 3.11 % of cells showed DNA damage. It might be due to the small percentage of cells undergoing apoptosis. In the test sample treated for breast cancer MCF-7 and lung cancer A549, around 16.50 % and 12.60 % of cells have shown DNA damage, whereas, in the case of standard drug cisplatin-treated for breast cancer MCF-7 and lung cancer A549, DNA damage was observed in 25.90 % and 13.70 % of cells, respectively (Figures S21 and S22).