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. 2023 Aug 2;13(8):289. doi: 10.1007/s13205-023-03705-w

Isolation and anticancer activity of quercetin from Acalypha indica L. against breast cancer cell lines MCF-7 and MDA-MB-231

Sudhakar Chekuri 1,#, Satyanarayana Swamy Vyshnava 2,3,#, Swarupa Lakshmi Somisetti 4, Sai Bindu Karamthote Cheniya 1, Chakradhar Gandu 5, Roja Rani Anupalli 1,✉
PMCID: PMC10397153  PMID: 37547624

Abstract

In this study, the active components of the plant were carefully extracted and identified using three solvent systems. After extraction, we used solvent systems to further purify the main flavonoid chemical constituent. As a result of our analytical strategy, which included HPLC analysis, MS/MS spectroscopic analysis, and NMR data-based constructions, quercetin was determined to be the main chemical constituent. Our study suggests the potential therapeutic advantages of quercetin, a compound found in the leaves of Acalypha indica, for treating breast cancer cell lines MCF-7 and MDA-MB-231. Our comparison of Quercetin to the regularly prescribed medicine Doxorubicin shows that it has the capacity to inhibit MCF-7 and MDA-MB-231 cells. Measurements of apoptosis and cell cycle phase showed this to be the case. Furthermore, a ladder that formed as a result of cellular damage brought on by ROS provided further proof of the drug's impact on DNA integrity. Notably, pro-apoptotic proteins displayed increased apoptosis activity in cells treated with quercetin. Given that it is extracted from plants and has less adverse effects than other compounds, quercetin is a viable option for further clinical study. The objective is to fight breast cancer, one of the most prevalent diseases in the world and a main cause of death for women. Thus, our research makes a significant addition to the ongoing search for potent, plant-based breast cancer treatments.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-023-03705-w.

Keywords: Acalypha indicia, Anti-cancer compound, Apoptosis, MCF-7, MDA-MB-231, Plant extract, Quercetin

Introduction

World cancer incidence and mortality estimate that 14.1 million people will be diagnosed with cancer in the upcoming years. 8.2 million people were estimated to die of cancer, while 32.6 million will live with cancer as a chronic related issue (Bertucci et al. 2019; Waks and Winer 2019). According to estimates, there will be 26 million new cancer diagnoses and 17 million cancer-related deaths by 2030 (Hassanpour and Dehghani 2017). Despite significant efforts, cancer remains the leading cause of death worldwide (Mortezaee et al. 2019). Additionally, novel synthetic chemotherapeutic medicines in clinical trials have not met expectations regardless of their actual development costs (Chandrasekar et al. 2018). The increasing number of cancer cases has resulted in the constant need for practical and cost-effective anti-cancer drugs (Hassanpour and Dehghani 2017). Compounds derived from plants have been used to cure human diseases since the dawn of medicine (Bryda and Stadnytska 2021). Due to their potential as cancer prevention and treatment agents, natural products have received much attention in the last 30 years (Gangola et al. 2017; Chavda et al. 2021). Further evidence shows that plant-derived compounds such as polyphenols (Ameer et al. 2017), flavonoids (Niranjan et al. 2011), lignin (Formica and Regelson 1995), alkaloids (Yun et al. 2021), terpenoids (Kamran et al. 2022), carotenoids (Ávila-Román et al. 2021), and other bioactive compounds with importance in cancer prevention and treatment (Roy et al. 2017; Azmir et al. 2013).

Around 60% of modern cancer treatments are natural products, with plants being the most significant source (Shoemaker et al. 2005). Medicinal plants are used in many countries worldwide to treat cancer (Huang et al. 2021). Currently, over 3000 plants have been identified as having anti-cancer properties globally (Yun et al. 2021). Plant-derived products are used for treatment by 10–40% of cancer patients worldwide, with a 50% rate among Asian patients (Kainsa et al. 2012). Next-generation plant-based bioactive compounds must have fewer adverse effects, are less expensive, and are more effective in the present situation. There has been much interest in biologically potential extracts and their therapeutic chemicals obtained from various medicinal plants in recent years (Kainsa et al. 2012; Mickymaray et al. 2016).

Several types of cancer cells were strongly inhibited by Quercetin, including those from the breast, lungs, stomach, ovarian, colorectal, and liver (Manach et al. 1995; Boots et al. 2007; Molani Gol and Kheirouri 2022). The anti-cancer effects of Quercetin ascribe to several mechanisms, including cell death, the blockage of angiogenesis, the blocking of P-gp channels, the reduction of oncogene expression, and the regulation of signal pathways (Li et al. 2016). Rheumatoid arthritis, ulcers, and other diseases are worsened by inflammation because it produces inflammatory mediators (Rauf et al. 2018). Following research, Quercetin exerts an anti-inflammatory effect by suppressing proinflammatory cytokines, ATP, and nuclear factor kappa B (IkB) binding sites (Kashyap et al. 2022; Becer and Vatansever 2022).

The plant Acalypha indica has medicinal properties that can benefit human health. Many cultures in Asia and Africa use A. indica as traditional medicine (Solomon et al. 2005; Govindarajan et al. 2008). There are several places in western and southern Africa where it can be found, including Ethiopia, Somalia, and other countries (Dineshkumar et al. 2010; Takle et al. 2011). There are medicinal plants in many countries in Asia, Europe, and South and North America (Teklani and Perera 2016). This plant's leaves, roots, and stems treat eye infections, respiratory issues, rheumatism, skin issues and lower blood sugar levels (Venkatachalam et al. 2017). Various methods are accessible to extract active components from A. indica. It is generally true that Soxhlet extraction has high efficiency and accuracy, but thermal stress may lead to the degradation of target photochemical components (Zahidin et al. 2017; Ninave and Patil 2022; De Castro and Priego-Capote 2010).

Novel therapies are needed to minimize the numerous complications associated with existing therapeutic drugs in treating breast cancer. Our hypothesis posits that the isolation of bioactive components from A. indica has the potential to serve as a viable therapeutic option for the treatment of breast cancer. Examining potential bioactive compounds, such as Quercetin, through systematic extraction methods, established cytotoxicity, and anti-tumour treatment methods for breast cancer cell lines include MDA-MB-231 and MCF-7.

Materials and methods

Chemicals

In our study, we purchased analytical grade solvents, including Methanol (CH3OH), Chloroform (CHCl3), Ethyl acetate (C4H8O2), n-Butanol (C4H10O), and Dichloromethane (CH2Cl2), were purchased from Loba Chemie Pvt. Ltd. Chemicals for phytochemical studies which include, 1, 1-diphenyl-2- picrylhydrazyl (DPPH), Vanillin (C8H8O3), Aluminium trichloride (AlCl3), Naphthyl ethylene diamine dichloride (C12H16Cl2N2), and tannic acid from Himedia. Hydrogen peroxide (H2O2) and Lead acetate (Pb(C2H3O2)2) were purchased from Finar Chemicals. For cell culture studies and immunomodulatory assay Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute Medium (RPMI- 1640), Penicillin (Pen), Streptomycin (Sm), Dimethyl sulfoxide (DMSO), and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) were purchased from Himedia. Fetal bovine serum (FBS) from Gibco. The chemical-based kits were purchased from Invitrogen.

Preparation of plant extract

The A. indica plant material was collected from the Central Institute of Medicinal and Aromatic Plants (CIMAP) in Hyderabad, India (BSI/DRC/2021–2022/Tech/Identification/287). Methanol, Ethyl acetate, and chloroform are used to extract 500 g of powdered leaf material in 1000 mL of organic solvent using Soxhlet extraction for 8 h (The polarity index for Methanol, Ethyl acetate and chloroform are 5.1, 4.5, and 4.0 respectively). Powdered leaves were dissolved in an organic solvent at 50 °C continuously stirring. Three extracts were filtered using 5 M Whatman paper, and all three extracts were evaporated at 40 °C using a vacuum rotary evaporator (Heidolph, Germany). After evaporation, the crude extract was kept in a desiccator. After extraction with three solvents, the residual residue was removed, and each dried crude extract was weighed. Physical properties and percentage yields of various extracts are documented.

Isolation and characterization of quercetin

The crude methanolic extract was considered a potential source of Quercetin due to its relative cytotoxicity. A crude methanolic leaf extract of A. indica was weighed and fractionated. Methanol fractions were collected, and aqueous methanol phases were evaporated under reduced pressure to form semisolids. In 200 mL of distilled water, the semisolid portion was dissolved and fractionated with aqueous methanol (MeOH), dichloromethane (DCM) and n-butanol (BuOH). For further analysis, the dried fractions were dissolved in methanol and stored in amber-coloured vials after fractionation. The concentration of extracts used in all experiments was 10 mg/mL.

The relative cytotoxicity of flavonoids, total phenols, triterpenoids, terpenes, and saponins from methanolic extracts of A. indica leaves was assessed. In order to quantify biomolecules, a defatted sample is essential. The extraction procedure (explained in Supplementary Information) was followed to prepare a defatted sample. Using a Soxhlation approach, 300 mL diethyl ether was dissolved in powdered material for 2 h.

GC–MS spectral studies

GC–MS analysis was performed later to examine the bio-molecular distribution of methanolic leaf extracts of A. indica. Perkin-Elmer (Auto System XL) Clarus 500 Gas Chromatograph equipped with a Turbomas 5.2 spectrometer and an Elite-1 (100% Dimethyl Polysiloxane) capillary column were used for GC–MS analysis. By utilizing the National Institute of Standards and Technology Mass Spectral information base (NIST-MS), discontinuity examples of mass spectra were contrasted and stored in the spectrometer information base. The percentage of each component was calculated based on the relative peak areas of each component in the chromatogram. The NIST library’s known and unknown components were compared (Mohan et al. 2012; Chekuri et al. 2016).

HPLC spectral studies

Characterization of methanolic aq was performed using HPLC. To validate the identity of individual compounds and verify the purity of fractions obtained after successive column purification of A. indica leaf extract. The HPLC system consists of two LC pumps (Perkin-Elmer Series 200 and a high-pressure mixer), an autosampler (Norwalk, CT) with a 10 L sample loop and Chromolith® Performance RP-18, and a Phenomenex column (100 × 4.6 mm, 3 mm particles). Thermo Scientific Chromeleon software edition 6.80 was used for data processing and analysis (Qin et al. 2021; Jackson Seukep et al. 2020).

HR-MS/MS spectrum

The high-resolution mass spectrometer was operated in positive ion mode and had the following requirements: capillary voltage, 2200 V; source temperature, 100 °C; desolation temperature, 350 °C; cone voltage, 30 V. Over a mass spectrum between 300  and 750 m/z, the spectra are scanned at 2.0 s per cycle. NIST's molecular profile database detects individual substances under common chromatographic conditions for standardized compounds. Results were interpreted and processed using Chromeleon Client version 6.80 (Thermo Scientific) (Ballesteros-Vivas et al. 2019).

Proton NMR

Proton NMR spectra of Quercetin were taken on a Bruker MSL 500 NMR spectrometer. Data were processed in MestReNova-11.0.3, and 1H-NMR spectra were compared with available research literature data (Martini et al. 2008).

Cell culture

Three tumour cell lines were selected to screen A. indica leaf extracts in MeOH, Ethyl acetate (EAc) and chloroform (Chl). Subcultures of the cell lines include A549 (lung adenocarcinoma), MCF-7 (breast adenocarcinoma), and A-375 (human melanoma cells) cell lines opted from the PGP Life Sciences, Hyderabad, TS. The cell line's initial source is the ATCC (American Type Culture Collection). A humidified atmosphere of 5% CO2 was maintained at 37 °C in 75 cm2 bottle-necked vented flasks (Corning) with DMEM. The cell lines were grown in DMEM with 10% FBS, 1% non-essential amino acids (NAA), 1% Pen (1000 U/mL), 1% Sm (1000 g/mL), and 1% amphotericin (Am) (250 U/mL). On 75 cm2 plastic flasks, cells were sub-cultivated at a density of 2.2 × 104 cells/cm2 using 0.25% trypsin-1 mM EDTA enzymatically. We have changed the medium every two days. Microscopic observation confirms cell confluence (80%).

To estimate the cytotoxicity with the MeOH fractions, 96-well tissue culture plates with clear bottoms, we plated and cultured MCF-7, MDA-MB-453, and MDA-MB-231 human breast adenocarcinoma cells. The number of cells was 7 × 103 per well. Various concentrations of aqueous (Aq.), dichloromethane (DCM), and n-butanol (n-BuOH) fractions of A. indica leave extracts were applied to the cells after 24 h of seeding, and the cells were incubated for 24 h at 37 °C, 5% CO2. The incubation medium was removed from all wells of a plate and washed three times with PBS. MTT reagent was made up in PBS medium to a final concentration of 0.5 mg/mL, then added to each well. Reagent volume should be adjusted based on cell culture volume. Optical density (OD) was measured for each well on an absorbance plate reader at 570 nm for 3 h at 37 °C until intracellular purple formazan crystals were visible (Azizi et al. 2017; Vyshnava et al. 2020a, 2022).

Cell cytotoxicity assay

The MTT Cell Viability (A549, MCF-7 and A-375 cell lines) assay provides a convenient, sensitive, quantitative, and dependable way to measure how many viable cells are in a culture. MTT assay measures cell viability, proliferation, and cytotoxicity by measuring metabolic activity. A homogeneous colorimetric assay transfers the tetrazolium salt MTT, a pale-yellow substance, to formazan, a purple compound. NADH/NADPH cofactors are present during cellular reduction, and only living cells can catalyze the reaction. The formazan substance is not soluble in water and is purple. Dissolving the resulting formazan in a solubilization buffer allows convenient quantification of substance formulation (Burton 2005). A substance's colour strength is directly proportional to the number of cells living in the culture at 550–620 nm. The increasing proliferation of cells corresponds to an increase in the signal, whereas decreasing proliferation of cells can indicate toxic compounds or suboptimal conditions. Under a microscope, intracellular purple formazan crystals were visible after 3 h at 37 °C. Each well was measured on an absorbance plate reader at 570 nm optical density (OD) (Mbaveng et al. 2018). In all experimentations of the cytotoxicity studies, we used 1% DMSO as the vehicle control.

Anti-cancer activity

Human breast cancer cells, including MCF-7, MDA-MB-453, and MDA-MB-231, were plated and cultured in DMEM of growth medium per well of a transparent flat bottom 96-well tissue culture plate (There were 7 × 103 cells per well). Following 24 h of seeding, the cells were exposed to incremental 0.1 to 50 µg/mL concentrations of Quercetin isolated from A. indica leaves and incubated at 37 °C with 5% CO2. After incubation, we removed the growth medium from all wells and washed them three times with 1 × PBS. Each well was treated with 15 µL of MTT reagent in PBS medium at a final concentration of 0.5 mg/mL. Reagent volume should be adjusted based on cell culture volume. After 3 h at 37 °C, intracellular purple formazan crystals could be seen under a microscope. At 570 nm optical density, each well was evaluated using an absorbance plate reader (OD).

MTT assay was used to determine Quercetin’s time-dependent cytotoxicity after incubation for 24, 48, and 72 h. In 96-well tissue culture plates with a flat bottom, we cultured human breast cancer cells MCF-7, MDA-MB-453, and MDA-MB-231. (Each well had 7 × 103 cells). After 24 h of seeding, cells were exposed to different concentrations of Quercetin made from Acalypha indica leaves, ranging from 0.1 to 50 mM solutions. They were incubated for 24, 48, and 72 h at 37 °C and 5% CO2. Incubated wells were washed with 1xPBS three times after removing the growth medium. In each well, 15 µL of MTT reagent was added in 1xPBS medium at a 0.5 mg/mL concentration. Depending on the cell culture volume, adjust the magnitude of the reagent. A solution of 10% acidified Sodium dodecyl sulphate (SDS-HCl) was added to the cells after 3 h at 37 °C to dissolve the purple formazan crystals formed within the cells (Kumar et al. 2018). The absorbance was measured at 570 nm for each well using an absorbance plate reader (Ghagane et al. 2017).

Apoptosis induction

The initial apoptotic effects were assessed using an Annexin V-FITC and PI kit (BD Biosciences, San Jose, CA, USA) as directed by the manufacturer. A six-well plate containing approximately 3 × 105 cells was plated the night before and given the night to attach. Cells were treated for 48 h with Quercitin at doses equal to the IC50, then trypsinized and resuspended in 1xPBS at a cell density of 1 × 106 per ml. After mixing the suspension with binding buffer, Annexin V-FITC, and propidium iodide (PI), it was incubated at room temperature for 20 more minutes in the dark. The binding buffer, a volume of 500 µL of sample volume, was treated. A BD FACS Calibur flow cytometer (BD BioSciences, USA) was used to analyze samples after vortexing (Abd Razak et al. 2019).

Apoptosis by DNA laddering assay

Apoptosis is characterized by irreversible DNA fragmentation, which happens before changes in plasma membrane permeability (prelytic DNA fragmentation). An endonuclease cleavage product of apoptosis can be visualized using DNA laddering. DNA is extracted from a lysed cell homogenate in this assay and then electrophoresed on an agarose gel (Alkahtani et al. 2022).

Analysis of cell cycle arrest

MCF-7 and MDA-MB-231 were stained with PI (Propidium iodide). Later, flow cytometry was used to assess the effect of Quercetin. We plated 5.3 × 105 cell lines and let them attach overnight. The medium was replaced with a fresh medium containing the desired amount of Quercetin. Incubated at 37 °C for 48 h, floating and adherent cells were collected, washed twice with cold 1xPBS, and fixed overnight with ice-cold 70% EtOH. The cells were then incubated for 30 min and treated with 80 mg/L RNase A and 50 mg/L PI. BD FACS Calibur flow cytometer was used to analyze stained cells. We reanalyzed the data using BD MultiSET (Prasad et al. 2020).

Western blot analysis

To further verify the apoptosis process in Quercetin treated cells through SDS-PAGE followed by western blotting to investigate the protein expression involved in apoptosis. SDS-PAGE was done under reducing conditions using polyacrylamide slab gels at 12.5%. A semidry electroblotting tank was used to transfer proteins from the gel to the PVDF membrane. After washing three times (each time 5 min) with wash buffer (0.05% Tween 20 in PBS), the membrane was soaked in the primary antibody (Abcam, USA) with a dilution of 1/1000 on the shaker for two hours, followed by three more washes with wash buffer. After 2 h of shaking, the membrane was incubated for 0.03% HRP conjugated secondary antibody (Abcam, USA, 3 mL in 10 mL wash buffer, at 1/5000 dilution). With wash buffer, the membrane was washed three times after discarding the secondary antibody. A radiography film was developed with an ECL substrate to establish the protein bands (Leng et al. 2018; Hargraves et al. 2016).

VEGFR-1 and Aurora kinase A Genes Expression

RT-PCR was performed to confirm Western blotting results of VEGFR-1 and Aurora kinase A expression. Therefore, using mRNA from MCF-7 and MDA-MB-231 cells, PCR was carried out on synthesized cDNAs. Electrophoresis through a 2% agarose gel followed by ethidium bromide (EtBr) staining visualized all RT-PCR products (Mohajeri et al. 2020).

Molecular docking and analysis

The structures of the compounds were sketched in ChemDraw and used as ligands. In Open Babel, nine SD (.sdf) files were converted to their corresponding three-dimensional (.pdb/.mol2) structures. The Gasteiger charges were added, and non-polar hydrogens were merged with AutoDock 4.2. Using the Research Collaboratory for Structural Bioinformatics (RCSB) and protein data bank (PDB), the 3D crystal structures of human Cyclin B (2JGZ), Cyclin D (2W96), Cyclin E (1W98), VEGFR (2VWE), IKKB (3BRV), GSK3 (4NM0), PI3 (7POP), BCL-2 (4C5D), and BCL-XL (7LH7) were retrieved, without complexed ligands. Hydrogens were removed from protein structures using AutoDock4.2, Gasteiger charges were calculated, polar hydrogens were added, and non-polar hydrogens merged using AutoDock4.2 (Rizvi et al. 2013). Both receptor and ligand structures were converted to PDBQT files using AutoDock4.2, which contain atomic charges, atom types, and topological information (rotatable bonds) for ligands. In addition to ensuring coverage of the binding site of the structure, a grid was used to ensure that the binding site was covered. AutoDock4.2 generated nine conformations of the ligand in complex with the receptor to perform docking simulations, which were then ranked (Vyshnava et al. 2020b).

Results and discussion

Isolation of crude extracts from Acalypha indica dried leaves

Using the phytochemical extraction from the A. indica leaves extract, we systematically observed Quercetin's effect on breast cancer cell lines. Standard procedures were followed to extract phytochemicals using various organic solvents. A indica leaves were allowed to settle for 24 to 72 h at a temperature closest to their near boiling points of organic solvents, as shown in Fig. 1a. After observing the dissociation of the plant compounds into the solvents Fig. 1b, we allowed the solvents to filter through a 45 μm filter and evaporate in a vacuum rotavapor. As shown in Table 1, the percentage yields of the organic solvents are shown, with MeOH showing the highest yield at around 21.51%, followed by semi-polar organic solvent, such as EAc, at 16.69%, and chlor, a non-polar organic solvent, with an estimated yield of 11.84%. (Fig. 1c).

Fig. 1.

Fig. 1

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a Soxhlet apparatus used for the plant leaf extraction for Quercetin b Structure of the Quercetin in 2d and 3d perspectives c Three solvent systems showed the percentage of yield for plant leaf extraction, where methanolic extraction shows the promising percentage of bioactive phytochemical compounds

Table 1.

Percentage of Yield in various organic solvent systems of Acalypha indica. L leaves

Solvent Wt. of dry leaf powder in (g) Wt. of Yield
(g)		 Yield
(%)
Methanol 500 107.59 21.518
Ethyl acetate 500 83.46 16.692
Chloroform 500 59.22 11.844
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Phytochemical analysis of the crude extracts

Based on the previous research reports, a qualitative analysis was carried out using standard chemical methods (see Supplementary Information methodology) to assess the nature of phytochemical compounds or secondary metabolites found in the crude extracts (Azmir et al. 2013; Popov et al. 2017; Bryda and Stadnytska 2021). MeOH crude extract of A. indica contains several preliminary phytochemicals, including alkaloids, saponins, terpenoids, flavonoids, tannins, steroids, glycosides, and phenols. However, a few chemical constituents have mild concentrations in the EAc and Chl extracts, as listed in Supplementary Information Table S1.

Cytotoxicity of crude extracts

Further doses of the organic crude extracts from 5 to 320 µg/mL were added to the adenocarcinoma cell lines to observe their relative composition and cytotoxic efficacy. We observed that methanolic crude extract shows the potential cytotoxicity at a comparable concentration of 160 µg/mL in MCF-7 cell lines, followed by A549 and A375 cell lines, as shown in Fig. 2a. In the case of the EAc extract, the data revealed a moderate amount of cytotoxicity compared with 40% less inhibition than the MeOH extract, as mentioned in Fig. 2b. The Chl extracts show relatively negligible cytotoxicity compared with the other two solvent systems, which is 80% lower, as shown in Fig. 2c. The relative IC50 values of the three solvent extracts indicated a lower dosage of methanolic extract when treated with the experimental cell lines of about 200 µg/mL. In contrast, IC50 values reported for ethyl acetate extract were mild to moderate about 400 µg/mL and the chloroform extracts showed higher IC50 values depicted in Fig. 2d.

Fig. 2.

Fig. 2

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Bar chart representation for cytotoxicity of Acalypha indica leaves organic solvent include the (a) Methanolic extracts (b) Ethyl acetate extracts (c) Chloroform extracts (d) respective IC50 values on adenocarcinoma cell lines including A549, MCF-7, and A375. (With a non-significant value of P > 0.05 (observed P value of 0.9863))

The first step in determining the crude extract activity of a plant is to identify its polar chemical constituents, which inhibit MCF-7 and two other cell lines when compared to ethyl acetate and chloroform extracts, which contain fewer polar chemical constituents with a non-significant value of P > 0.05 (observed P value of 0.9863). These results agree with previous research observations regarding the phytochemical composition of methanolic and ethyl acetate extracts of A. indica, which suppress cancer cells (Zahidin et al. 2017; Suresh et al. 2021; Sophia et al. 2022). By analysing the phytochemical components of the crude methanolic extract, we isolated Quercetin as the molecule of interest. We further quantified crude methanol extract based on gravimetric measurements reported earlier (Raaman 2006; Kokate 2001).

GC–MS analysis of the methanolic extract

Based on the gravimetric data, the methanolic extraction yields abundant in flavonoids, phenolics, triterpenoids, tannins, and saponins are show in Fig. 3a. Figure 3b shows the relative spectral peak area in the GC–MS chromatogram of methanolic extract. Table 2 lists isolated phytochemical compounds corresponding to the NIST-MS library and the compounds previously reported with known functions mentioned in the Ruslan group, which were identified as antibacterial, anti-cancer, and anti-inflammatory compounds (Ruslan 2015). In contrast, Quercetin was observed in the spectrum at 32.19 min retention time, our compound of interest obtained with fewer functional applications. Previous research reports suggest that the anti-cancer properties of Quercetin compound are relatively for fewer cancers (Murakami et al. 2008; Hashemzaei et al. 2017; Rauf et al. 2018; Erdoğan et al. 2022; Becer and Vatansever 2022). Based on this experimental observation, we further proceeded to isolate the Quercetin in its pure form based on fractionation with organic solvents based on the polarity.

Fig. 3.

Fig. 3

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a Gravimetric content for bioactive compounds from the methanolic extraction, where flavonoids are dominant (b) GC–MS spectrum of methanolic leaf bioactive phytochemical content in flavonoids, where the observed presence of the Quercetin at 32.19 Retention time (Rt) of the spectrum

Table 2.

GC–MS spectrum of methanolic leaf extract of Acalypha indica, concerning various organic contents based on the spectral peaks

S. No *Rt Name of the compound Mol. Formula Mol.wt
(g/Mol)		 Peak Area
(%)
1 9.76 4-C-methyl-myo-inositol C7H14O6 194.22 15.85
2 11.92 Proline,3,4-didehydro C5H7NO2 113.67 100.00
3 15.06 3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran4-one C6H8O4 144.09 18.78
4 16.34 Cysteine C3H7NO2S 121.16 17.49
5 17.97 Ellagic acid C14H6O8 302.19 56.92
6 18.18 Diethyl phthalate C12H14O4 222.23 14.02
7 19.16 1H-Pyrrole-2,5-dione,1- ethenyl C6H7NO2 123.42 35.71
8 19.95 Chebulic acid C14H12O11 356.23 48.81
9 21.44 Gallic acid C7H6O5 170.12 39.24
10 22.84 4-Amino-3- methoxypyrazolo[3,4- d]pyrimidine, C6H7N5O 165.29 35.28
11 23.03 Propanenitrile,3-(5- diethylamino-1- methoxy3-pentynyloxy) C13H22N2O 223.16 13.91
12 24.89 Didodecyl phthalate C32H54O4 502.14 47.65
13 26.04 Trifluoromethyl t-butyl disulfide C5H9S2F3 190.21 33.67
14 27.18 Ethyl pentanate C10H20O2 172.48 46.12
15 28.15 Ethyl decanate C12H24O2 200.31 55.37
16 28.42 Rutin C27H30O16 610.51 63.63
17 31.02 Kaempfeorl C15H10O6 286.23 89.07
18 31.14 Phytol C20H40O 296.18 89.34
19 32.19 Quercetin C15H10O7 302.23 91.06
20 33.27 Squalene C30H5O 410.57 78.17
21 35.06 3,8-Nanodiene-2-one C9H14O 138.24 76.20
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*Rt retention time

Methanolic fraction extraction

Approximately equal quantities of crude methanolic extract were weighed and fractioned into three portions, dissolved in methanol and evaporated under reduced pressure to form a semisolid. The induvial fraction was made into methanol, dichloromethane (DCM) and n-butanol fractions. The dried fractions are collected and stored at ambient temperatures.

Cytotoxicity of the methanolic fraction

Further cytotoxicity was evaluated by comparing the methanolic crude extract fractionation to the individual cancerous cells originating from breast cancers. With a steady state increase in the concentration of the solvent fractions, the aqueous fraction of plant extract showed maximum toxicity towards MDA-MB-231 cells with 99% inhibition, as shown in Fig. 4a. A similar trend regarding the doxorubicin treatment for MCF-7 and MDA-MB-453 cells as shown in Fig. 4b, 50% of the relative toxicity was observed in the DCM fraction, and similar trends were observed across all cell lines. However, the observations in the n-butanol fractions show no significant cytotoxicity, which may be negligible, with similar trends in all the treated cell lines making up less than 20%, as shown in Fig. 4c. Regarding the DCM, the IC50 of the aqueous fraction is approximately 200 µg/mL, whereas the IC50 of the n-butanol is > 200 µg/mL with a non-significant value of P > 0.05 (observed P value of 0.8821). Based on the observation, we confirmed that the aqueous methanolic fraction contained most of the phytochemical to be isolated, which was further refined and chemically analyzed.

Fig. 4.

Fig. 4

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Bar chart representation for cytotoxicity of Acalypha indica leaves in a solvent extract with (a) Aqueous fraction (b) Di-chloromethane fraction (c) n-Butanol fraction (d) respective to IC50 values MCF-7, MDA-MB-453, and MDA-MB-231 cells. (With a non-significant value of P > 0.05 (observed P value of 0.8821))

Colum chromatography and HPLC for the methanolic extract

Aqueous methanolic extracts were purified in the first instance using activated silica gel in conjunction with eluting solvents and various solvent systems, as shown in the Supplementary information in Table S2. As a result of starting with the corresponding fractions with the solvent systems, it is possible to estimate the gravimetric percentage of yield. Methanol is the most promising eluent for further experimentation, with a maximum harvest of 27.39% at 17.69 mg mass weight, compared to 50 mg mass weight in the initial aqueous methanolic fraction in Fig. 5.

Fig. 5.

Fig. 5

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Percentage of yield for the various organic solvent systems to be executed for the aqueous methanolic fractions for the column chromatography, where methanol shows better for the extraction fractions

Following the Column mentioned above chromatography purification, the methanolic aqueous fraction was subjected to HPLC for further purification. As part of the analysis, standards are purchased for Quercetin for HPLC runs. As soon as the peaks were obtained, Quercetin was identified at 22.41 min retention time (Rt), all of which were analyzed through Fig. 6a. Based on the peak Rt, which includes the peak height and the peak breadth, Quercetin was identified for purity and quality, as represented in Fig. 6b, including peak 22.45 min applicable to Quercetin. Figure 6c shows that the MS/MS spectral data are used to analyze the HPLC purified fractions and determine their structural characteristics. MS/MS spectrophotometers run samples through to elute the data based on the NIST database. Figure 6c depicts the peaks obtained from the 302.04 m/z peaks for Quercetin, consistent with the previous research reports (Pinho et al. 2014; Jirge et al. 2014; Niranjan et al. 2011; Manach et al. 1995).

Fig. 6.

Fig. 6

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Isolation and identification of the compound of interest through HPLC runs for the Column purified aqueous methanolic fraction of Acalypha indica leaves extract, where with a structural correlation of (a) Commercially available compounds (b) Quercetin (c) MS/MS spectral analysis of the Quercetin from the HPLC based isolates, where the MS/MS peak shows the (ESI, M +): 302.04; found 302.04 m/z

NMR for the methanolic extract

We correlated the structural data sets from HPLC fractions with the NMR data sets, as shown in Fig. 7, to generate the respective constructs for Quercetin. The 1H protonated spectra of Quercetin exhibit chemical shifts of 1H NMR (400 MHz, CDCl3): δ 12.6 (s, 1H), 10.7 (s, 1H), 9.3 (m, 2H), 8.6 (s, 1H), 7.3 (m, 1H), 7.1 (s, 1H), 6.6 (m, 1H), 6.2 (m, 1H), 6.1 (m, 1H). Based on these data sets, the NMR analysis for plant-based extracts is consistent with earlier research (Wiese et al. 2013; Chandradevan et al. 2020).

Fig. 7.

Fig. 7

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1H NMR spectral data of Quercetin (400 MHz, CDCl3) [.1H NMR (400 MHz, CDCl3): δ 12.6 (s, 1H), 10.7 (s, 1H), 9.3 (m, 2H), 8.6 (s, 1H), 7.3 (m, 1H), 7.1 (s, 1H), 6.6 (m, 1H), 6.2 (m, 1H), 6.1 (m, 1H)]

Cytotoxicity of the quercetin

Quercetin isolated from sequential extraction has potent anti-cancer activity, and the aqueous methanolic fraction, compared to the other molecules identified in this study, had a lower IC50 value in inhibiting MCF-7, MDA-MB-453 and MDA-MB-231. According to Fig. 8a, MCF-7 cells treated with Quercetin for 24 h, 48 h and 72 h demonstrated a gradual increase in cytotoxicity with the different concentrations of Quercetin, ranging from 0.1 to 50 µg/mL. The bar chart Fig. 8a representations show similar trends, with no significant change in cytotoxicity in MCF-7 cell lines, which is 99% of cellular inhibition based on exposure time. MDA-MD-453 and MDA-MD-231 show similar trends as shown in Fig. 8(b, c), respectively, which indicate that inhibition at higher concentrations remains over 90% based on their time and dosage of Quercetin. As shown in Fig. 8d, the Quercetin's IC50 values against MCF-7, MDA-MB-453, and MDA-MB-231 cells lines are relative, where the Quercetin treated with MDA-MB-231 for the 72 h showed a lower IC50 value at 5 µg/mL compared with the 24 h and 48 h periods of exposure, while the MCF-7 requires 10 µg/mL and the MDA-MB-453 requires > 20 µg/mL.

Fig. 8.

Fig. 8

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Bar graph representation of IC50 values of Quercetin against (a) MCF-7 (b) MDA-MB-453 and (c) MDA-MB231 cell lines treated for a period of 24, 48 and 72 h of treatment, (d) IC50 values. (With a non-significant value of P > 0.05 (observed P value of 0.7136))

Quercetin appeared to be a potential inhibitor for the MDA-MB-231 cancer cell lines with lower IC50 values for different time slots and acting as a gradual inhibitor or death for the cancer cells as the time increased with a non-significant value of P > 0.05 (observed P value of 0.7136). The first study looked at the MDA-MB-231 cell line, which was not discussed in previous studies.

Cell morphology studies with quercetin compounds

The molecular analysis and biocompatible applications of the isolated fractions from Acalypha indicia leaves, the compound of interest, Quercetin, was further examined for cellular morphological studies. The cytotoxicity and morphological characteristics of MCF-7 and MDA-MB-231 cells were studied by exposing these cell lines to Quercetin for 24 h, 48 h, and 72 h as Doxorubicin at their IC50 value concentrations. Quercetin displays significant inhibitions with damaged cell borders and osteolytic appearance. Based on the observations in Fig. 9a, the MCF-7 and Fig. 9b, the MDA-MB-231 cell lines showed confluency of 20–40% and damage to cell borders when exposed to Quercetin and Doxorubicin for 24 h to 72 h. As we confirmed the change in the cellular morphology, i.e., damaging the cellular integral structure after treatment with Quercetin for the respective cell lines, we further assessed the stages of cellular death, also known as apoptosis; this activity was studied with the flow cytometric technique.

Fig. 9.

Fig. 9

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The morphological characteristics of the (a) MCF-7, (b) MDA-MB-231 when treated with the Quercetin at a concentration corresponding to their IC50 dosages and the changes are observed for a period of 24, 48, and 72 h of treatment

Apoptosis studies with quercetin

A concentration of Quercetin at the IC50 value was applied for 48 h to MCF-7 and MDA-MB-231 cells in a six-well microtiter plate at a density of about 3 × 105 cells per well. In both treated and untreated cell lines, cells are harvested and incubated with Annexin V-FITC and PI, followed by flow cytometry analysis to determine whether they have displayed apoptotic activity. As shown in Fig. 10a, MCF-7 showed that 62.95% of cells were in the early stages of apoptosis, followed by 31.56% intact cells and 2.43% necrotic cells. Observing Fig. 10b, we watched the 12.46% unchanged cell profile in a culture of MDA-MB-231 that has not been treated, while in the treated part of the culture, 79.81% of the cells are at the early apoptotic stage, as indicated by the frequency of the cells distributed with the FITC-Annexin V uptake. The most effective suppressed cell line for Quercetin treatment is MDA-MB-231. Based on these results, we further investigated the genomic DNA constituents in cells treated with Quercetin was based on these results.

Fig. 10.

Fig. 10

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Apoptosis study of the cell lines showing the control and treated with Quercetin (a) MCF-7, (b) MDA-MB-231 cell lines

DNA ladder studies with Quercetin

MCF-7 and MDA-MB-231 cellular DNA electrophoretic gel images depict the DNA ladder formation as shown in Fig. 11a, b. The comparison of gels showed that DNA ladders formed in the gels of MDA-MB-231 rather than MCF-7 as the concentration of Doxorubicin increased the number of DNA fragments. Quercetin, thus, may be involved in DNA damage, directly engaging cellular infrastructures and stimulating ROS-mediated cell membrane damage cascades to cause apoptosis.

Fig. 11.

Fig. 11

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DNA ladder formation when cells, including MDA-MB-231 and MCF-7 cells, are treated with Quercetin with various concentrations relative to its IC50 dosage

Cell cycle phases studies with Quercetin

A cell cycle study was conducted with the MCF-7 and MDA-MB-231 cell lines treated with Quercetin. MCF-7 and MDA-MB-231 graphical peaks show time-dependent percentages of cell cycle generations in Fig. 12a, b. In the MCF-7 and MDA-MB-231 cell lines, most cells are in the G1 phase, which is evident even though the time extends from 24 to 72 h. The Quercetin concentration increased from the IC50 to double the concentration of the IC50 values. When comparing the detailed analyses of the cell cycle phases for the MCF-7 cells for a while of 24 h to 72 h, in these cells, we found an increase in the percentage of cell distribution in the G1 phase when compared to control to treated samples. The same trend was observed in 42 h and 72 h for the G1-Phase, while the G2/M, S, and sub-G1 phases were consistent in the control sample rather than the treated sample, suggesting that the MCF-7.

Fig. 12.

Fig. 12

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Cell cycle phases of (a) MCF-7 and (b) MDA-MB-231 breast cancer cells treated with purified Quercetin compound for 24 h Cell cycle phases of MCF-7 and MDA-MB-231 breast cancer cells treated with purified Quercetin compound for 48 h

In contrast to MCF-7 cells, the MDA-MB-231 cell lines showed similar distributions when treated and untreated. Inhibition rates increased from 0.5 to 2 times with an increase in the IC50 dose, indicating that Quercetin inhibited cells at the G1 phase more efficiently than the MCF-7 cells, which showed a slightly slower inhibition rate. In contrast, MDA-MB-231 cell lines show dramatic reductions in Sub G1, S, and G2/M phased compared to untreated controls. The MDA-MB-231 showed a faster growth inhibition when treated with Quercetin than the MCF-7. Our observation concerning the time and concertation dependent of Quercetin was distinct to represent a not published data sets. Our study would be novel in approaching the plant-based extracts that will be leading sources for the treatment of cancer and related disorders.

Western blotting studies with Quercetin

Our approach was on the right track in understanding the cellular events for the treatment and application of Quercetin; now we approach the molecular level, including confirming the apoptosis with a base cellular expression of the proteins for apoptosis which will be caspase systems viz., procaspase-9, -8, -7, -3 which are best studied in the previous reports (Movsesyan et al. 2002; Fearnhead et al. 1998; Cui et al. 2007), Cleaved PARP (Bressenot et al. 2009; Casao et al. 2015), Bcl-2 (Dai et al. 2016; Vogler 2012), Bax (PORĘBSKA et al. 2006), VEGFR1 (Zhang et al. 2010), Aurora Kinase-A (Katayama et al. 2012) and standard cell protein β-actin (Lei et al. 2017; Khan et al. 2017). Figure 13 shows the western blot data for expressed proteins when treated with the Quercetin IC50 value concentration; all the pro-caspases, including pro-caspase 9,8,7 and 3, are expressed in both MCF-7, and MDA-MB-231 cell lines, which represents the activation of the apoptosis activity, along with the caspases, Bcl-2, cleaved PARP, Bax, VEGFR-1 and Aurora Kinase A was expressed relatively depicting the activity of the apoptosis activation, the cell viability was shown with the standard expression of the β-actin protein in the respective cell lines.

Fig. 13.

Fig. 13

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Western blot data sets of the pro-apoptotic protein’s expression in MCF-7 and MDA-MB-231 cell lines when treated with the Quercetin

Aurora Kinase A and VEGFR-1 studies with Quercetin

Similarly, the relative intensities of the expressed protein and controls for MCF-7 and MDA-MB-231 the normalized intensities are shown in Fig. 14. Comparing MCF-7 with MDA-MB-231, PARP, Bax, Bcl-2, and VEGFR-1 are expressed at a significant level. As a further step, protein expression was analyzed on the MCF-7 and MDA-MB-231 cell lines with and without Quercetin and control treatment, according to IC50 values. These results are compared with the viability of the cell lines with high expression of the β-actin protein. For the control, untreated cells, and treated cells, our analysis shows the augmented incremental expression of VEGFR-1 and Aurora Kinase A is higher in the MDA-MB-231 cells in comparison-to-comparison MCF-7 cells.

Fig. 14.

Fig. 14

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VEGFR-1, Aurora Kinase-A, and β-Actin protein expression analysis of the MCF-7 and MDA-MB-231 treated with the Quercetin in IC50 dosage

RT PCR studies with quercetin

We performed RT-PCR with the appropriate VEGFR-1 and Aurora Kinase -A genes primers to understand their expression in cells treated with the IC50 Quercetin concentration. In addition, after cell harvesting for the mRNA from the individual cell lines, concerning the treated and untreated Quercetin, RT-PCR was undergone to know the expression of the VEGFR-1 and Aurora kinase respective to the standard GAPDH as shown in the figure for the DNA gel electrophoretic gel Fig. 15, the appearance of the gels with 234 bp and 196 bp based for the respective cell lines include MCF-7 and MDA-MB-231 which are consistent with the previous results (Manikandan et al. 2010; Bakhashab et al. 2018), which refers the active formation of the apoptotic gene express for the Quercetin treated samples. The relative expressions of the VEGFR-1 and Aurora kinase A with the standard of the GADPH gene levels reveal the apoptotic genes' active formation.

Fig. 15.

Fig. 15

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RT-PCR-based VEGFR-1 and Aurora kinase-A genes expression studies when Quercetin treated on MCF-7 and MDA-MB-231

Molecular docking analysis

Among all Quercetin and apoptosis–anti–apoptosis complexes, the minimum binding energy was observed with BCL-XL complex with Binding affinity (ΔG) maxima of − 8.24 kcal/Mol with inhibition constant (Ki) of 916.33 nM followed by PI3 protein about ΔG − 7.97 kcal/Mol with Ki of 1.45 µM. Approximately nine amino acid residues interacted with the ligand molecule, including THR158, HIS147, GLU189, ILE190, GLY336, VAL337, ALA338, GLU340, and ASP341 from the receptor of interest with the Quercetin which is shown in Table 3. THR, HIS, GLU, ILE, GLY, VAL, ALA, GLU, ASP, and TYR interacted with strong hydrogen bonds with Quercetin. Alkyl and van der Waals interaction was another interaction observed with CYS, LEU, ALA, LEU, and LYS bonded with Quercetin via an alkyl group, as shown in Fig. 16a,i. Along with the weak interactions, there were four distinct types of bond interaction: THR and LEU exhibited pi-sigma interaction with the Quercetin. A typical carbon-hydrogen bond was created by ASP, LYS, VAL, LEU, LEU, LEU, and MET and reacted with the ligand by interacting with alkyl bonds. The van der Waal forces of the remaining residues reinforced these interactions.

Table 3.

Molecular docking of Quercetin with the function apoptosis and anti-apoptosis proteins

Receptor Ligand Binding Affinity
(Kcal/Mol)		 Inhibition Constant
(Ki)		 Cluster RMSD Number
H-bonds		 Active amino acids
Cyclin B Quercetin − 6.86 9.34 µM 1.22 6 THR158; HIS147; GLU189; ILE190; GLY336; VAL337; ALA338; GLU340; ASP341
Cyclin D Quercetin − 6.89 8.86 µM 2.10 7 CYS38; ALA39; ARG87; LEU148;LYS149; ARG61; GLU64
Cyclin E Quercetin − 6.30 23.91 µM 1.57 3 THR158; ARG217; LEU219; GLU260; MET261; TYR262
VEGFR Quercetin − 6.20 28.61 µM 1.89 5 GLY59; CYS61; GLU67; CYS68
IKKB Quercetin − 6.80 10.30 µM 2.93 3 ILE65; SER68; VAL709; ALA712; HIS713; CYS716; ALA64
GSK3 Quercetin − 7.32 4.31 µM 3.10 2 ILE62; ALA83; ASP133; ARG141; LEU188
PI3 Quercetin − 7.97 1.45 µM 0.63 2 LYS123; ARG460; ASN462; PRO463; PRO658; ALA661; LEU662
BCL-2 Quercetin − 5.86 50.32 µM 3.18 4 SER4; ARG6; GLU7; VAL10; TRP24; ALA84; LYS87
BCL-XL Quercetin − 8.24 916.33 nM 0.37 5 ALA93; TYR101; GLY138; VAL141; ASP133; ARG139
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Fig. 16.

Fig. 16

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3D representation of molecular docking of the Quercetin with the function apoptosis and anti-apoptosis proteins include (a) Cyclin B (b) Cyclin D (c) Cyclin E (d) VEGFR (e) IKKB (f) GSK3 (g) PI3 (h) BCL-2 and (i) BCL-XL

Conclusions

Our research showed a therapeutic drug made from the leaves of the Acalypha indicia plant that may treat breast cancer cell lines, including MCF-7 and MDA-MB-231. We systematically separated and identified the component of interest using the three solvent systems for crude extracts. The plant extracts were then processed using solvent systems for flavonoid chemical component purification. The identifying chemical of interest was Quercetin, deduced using HPLC analysis, HR-MS/MS spectroscopic analysis, and NMR data-based constructs to infer purity and structural correlations. Compared to the standard drug Doxorubicin, Quercetin showed potential inhibitions in MCF-7 and MDA-MB-231 cells, as evaluated by apoptosis and cell cycle phase measurements. The DNA integrity was demonstrated by forming a ladder because of ROS-induced cell damage. Cells treated with Quercetin, pro-apoptotic proteins, revealed enhanced apoptosis activity. Overall research, we concluded that Quercetin would be many drugs of interest in treating breast cancer cell linage.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2154 KB) (2.1MB, docx)

Acknowledgements

The authors would like to thank for the support from the Department of Genetics, Osmania University, for providing the resources and moral support to complete this research.

Author contributions

SC, and SSV equally contributed in this research and involved in Conceptualization, Methodology, Investigation, Formal analysis, Writing—Original Draft, SSV managed to curate the software applications, Validation, Formal analysis, Investigation, Resources was provided by Ms. SLS, and Prof. RRA, Data Curation was done by Ms. SBKC, and Mr. CG and finally Supervision, Project administration, Writing—Review and Editing of this research was supported by Prof. RRA.

Funding

This research received no specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The complete data is provided in supporting information.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Sudhakar Chekuri and Satyanarayana Swamy Vyshnava have contributed equally to this work.

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