Induction of apoptosis through downregulation of anti-apoptotic genes bcl-2 and Nucleolin in MCF-7 cells by Cleome gynandra leaf fractions containing Tricin, Pinocembrin, and Acacetin

Induction of apoptosis through downregulation of anti-apoptotic genes bcl-2 and Nucleolin in MCF-7 cells by Cleome gynandra leaf fractions containing Tricin, Pinocembrin, and Acacetin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Induction of apoptosis through downregulation of anti-apoptotic genes bcl-2 and Nucleolin in MCF-7 cells by Cleome gynandra leaf fractions containing Tricin, Pinocembrin, and Acacetin Saravanan Renuka, Sridharan Sriram, Ramalingam Sivakumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5755739/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigates the anti-proliferative effect of Cleome gynandra leaf fractions on MCF 7 cells. Chloroform extract of Cleome gynandra leaves (CCG) was subjected to column chromatographic separation with increasing polarity of solvents. The total fractions (36) collected were subjected to TLC analysis and pooled into three fractions (F1-F3) based on their R f value. Further, these fractions were screened for anti-proliferative and apoptotic effects on MCF7 cells. The IC 50 values for the CCG fractions (F1-F3) were 82.78, 93.69, and 200µg/mL, respectively. Of these fractions, F1 demonstrated a remarkable cytotoxic effect and was subsequently analyzed using LC-MS/MS. For the first time, the plant F1 is found to contain Tricin (a methylated flavone), Pinocembrin (a flavone), 4-hydroxy-2,3,4,6-tetramethoxychalcone (an aromatic ketone), Acacetin (a methylated flavone), and Cinnamic acid. The expression of bax, CYCS, bcl-2, and nucleolin was analyzed using RT-PCR. The MCF 7 cells treated with the F1 showed downregulation of anti-apoptotic genes bcl-2 and nucleolin and upregulation of bax and CYCS. Thus, the induction of apoptosis is mediated through the downregulation of the anti-apoptotic genes. Further, an in silico approach was employed to evaluate the binding affinity of the identified compounds with the Bax, nucleolin, and bcl-2 proteins. It was observed that Acacetin exhibited a stronger binding affinity with bax, Tricin, Nucleolin, and Pinocembrin with bcl-2. Based on this study, Tricin, Pinocembrin, Chalcones, and Cinnamic acid could target the apoptotic and anti-apoptotic genes studied. Biological sciences/Biochemistry Biological sciences/Cancer Cleome gynandra MCF-7 Column chromatography Apoptosis Nucleolin and bcl-2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Breast cancer is the most widespread type of cancer in women worldwide [ 1 ]. It is a malignant tumor commonly found among Indian women and a significant cause of mortality [ 2 ]. Female breast cancer proportion of new cases is 2.3 million among all cancer cases, and 6.9 is the mortality proportion of total mortality or mortality due to cancer [ 3 ]. It is a heterogeneous cell-cycle deregulatory disorder whose behavior is determined by molecular characteristics. Despite advances in medical screening, therapeutics, and effective prognosis, cancer still poses a broad threat to human health [ 4 ]. Emerging evidence from clinical and epidemiological studies suggests that genetic, environmental, and other pathophysiological conditions are the primary causative agents for cancer [ 5 ]. Breast cancer is often unpredictable, causing premature mortality due to the inadequate efficacy of existing therapeutics. Usually, it is associated with severe side effects. In the past, breast cancer was widespread in industrialized areas. The disease is spread over a vast geographical region and is present in every economic stratum. Because of the molecular variation and complexity underlying breast cancer occurrence, its treatment using chemotherapy and radiotherapy is very complicated and often leads to undesirable side effects. Plants and their extracts have been used to treat almost every disease for centuries, and breast cancer is no exception. Herbal medicines contain many chemical compounds, which include alkaloids, flavonoids, glycosides, saponins, resins, and terpenoids. There are complex herbal polyphenols present in the human diet. These compounds counterattack the tumor cells through apoptotic mechanisms [ 6 , 7 ]. Many plants and their constituents have been found to have promising anti-cancerous properties against breast cancer cells in both in vivo and in vitro models over the past ten years. Because there aren't enough randomized clinical trials, their therapeutic benefits in the treatment of breast cancer are still disputed. A popular approach for identifying potent anticancer drugs is isolating compounds based on bioactivity. Plants are a new, reliable source of anticancer compounds. Numerous essential anticancer medications are now generated from natural sources, including analogs, etoposide analogs, Camptothecin, Indicine N-oxide, Paclitaxel, Topotecan, Vinblastine, and Vincristine [ 8 ]. Aesculusindica, Garcinia mangostana, Pfaffia paniculata, Sapiumellipticum, Solanum nigrum, Artemisia vulgaris , and Vernonia amygdalina [ 9 ] are good examples of valuable medicinal plants in cancer treatments. These contain phytocompounds with naturally occurring antioxidant and anticancer properties and are known to inhibit or kill carcinogenic cells [ 10 ]. Cleomaceae as a source of anticancer plants. Traditional medicines play a significant role in India and are frequently the most readily available and cost-effective therapeutic choice for people. The naturally occurring species Cleome gynandra , which belongs to the Cleomaceae family, is often found in and around the Thanjavur region of Tamil Nadu, India. In many African nations, Cleome gynandra is a popular herb and widely dispersed plant located mainly in tropical and subtropical areas (Fig. 1 ). Flowers of cleome can be white or yellow. In many places, this herb's seeds and leaves are employed in traditional system of medicine [ 11 ]. Leaf sap has curative powers and is an analgesic to relieve pain from migraines, earaches, etc . The ears, nose, and eyes are stuffed with fresh leaves. Additionally, epileptic fits are treated using it. A combination of leaves or roots is used to alleviate a broad range of ailments, especially severe threadworm infection, conjunctivitis, stomachaches, chest pains, and arthritis can be treated using these leaves. Rheumatism, neuralgia, and headaches can be treated by rubbing them on the affected areas or applying them as a poultice [ 11 ]. Cleome gynandra is frequently used to treat rheumatoid arthritis, malaria, gonorrhea, and diarrhea. It is employed as an anti-helminthic and rubefacient in India. Scientific evidence has been gathered to support its analgesic, anti-inflammatory, anticancer, and antipyretic effects [ 11 , 12 , 13 ]. Because evolutionarily related plant species usually exhibit a high degree of similarity in the types of secondary metabolites and biological activities, these plant characteristics make it a suitable target for anticancer screening. This study is dedicated to reporting the potential of some novel herbal compounds, such as Acacetin, Chalcones, Cinnamic acid, Pinocembrin, and Tricin, to prevent and treat breast cancer. The presence of identified compounds, a well-known antioxidant and anticancer agent, has been reported for the first time in Cleome gynandra . Several studies showed the overexpression of nucleolin and bcl2, the anti-apoptotic genes, in different forms of cancer. Nucleolin has been correlated with increased stability of bcl2 mRNA and helps cancer cells avoid apoptosis (44). Hence, an attempt has been made to assess the cytotoxic effect of Cleome gynandra fractions on MCF7 cells. Materials and Methods 2.1. Chemicals Reagents used in the experiments: medium and Fetal Bovine Serum (FBS) from (GIBCO, USA) and silica gel 100–200 Mesh (for column chromatography) from Fluka Chemie GmbH (Gesellschaft mit beschränkter Haftung, Buchs, Switzerland) were purchased. All chemicals used in the studies were of reagent grade unless otherwise stated and procured from India. TRI Reagent (Sigma- Aldrich Inc. USA), primers & master mix (Quiagen). Unless otherwise noted, all the chemicals used in the studies were of reagent grade and procured from India (Sigma India). 2.2. Experimental setup The leaves of Cleome gynandra L. were collected from Thanjavur (Nov 2018), identified and authenticated by Botanist, CARISM (Centre for Advanced Research in Indian System of Medicine), SASTRA Deemed to be University, Thanjavur, and the herbarium was submitted to CARISM. The same was approved by Dr.N.Ravichandran, Botanist, SASTRA Deemed to be University, CARISM. Identification was also further confirmed compared to specimens recognized at Rapinat Herbarium (RHT, 172), St. Joseph’s College, Trichy, Tamil Nadu, India. Methods were carried out by relevant guidelines and regulations of CARISM, SASTRA Deemed to be University, Thanjavur (Voucher Number – CARISM00177). 2.2.1 Preparation of plant extract Fresh leaves of Cleome gynandra were washed, dried in an oven, and crushed to a coarse powder. About 100 gm of dried sample was macerated with 150 mL of chloroform and kept at room temperature (Overnight) in a shaker. The extract was collected and dried at 40ºC, allowed to lyophilize using a freeze dryer and stored at 4ºC until use. The crude chloroform extract (5gm) was fractioned by column chromatography and further analyzed by identifying molecules using LC-MS/MS (UHPLC Dionex C18 RP Acclaim 120 Å, 2.1 × 150 mm, 3.0 µm column, USA and MSMS Bruker Q-II TOF)[ 14 ]. 2.3. Crude extract fractionation Two grams of C. gynandra were lyophilized. The sample was dissolved in 5 mL of chloroform and was then allowed to dry with the mixing of silica gel G (60–100 mesh). A slurry was prepared and then packed to the column (15 cm) with the help of n-hexane (100%), followed by the mixtures of chloroform, ethyl acetate, and ethanol (90:10, 80:20, 70:30, and finally 50:50) of increasing polarity to obtain fractions. About 36 fractions of CCG were eluted with different solvents with increasing polarity. All the collected fractions were subjected to TLC, and R f values were calculated. All 36 CCG fractions were pooled into three fractions (F1-F3) based on their R f values. 2.4. MTT assay A cytotoxic assay on MCF-7 with CCG was performed using the MTT assay [ 15 ]. MTT is a tetrazolium salt. The enzyme succinate-dehydrogenase reduces tetrazolium into insoluble formazan, purple. The amount of formazan reflects the number of viable cells present. Percentage of cell inhibition (%) = \(\:\frac{100-\text{A}\text{b}\text{s}\text{o}\text{r}\text{p}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}{\:\text{a}\text{b}\text{s}\text{o}\text{r}\text{p}\text{t}\text{i}\text{o}\text{n}\:\text{o}\text{f}\:\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\) x 100 2.5. In vitro anticancer activity The human breast adenocarcinoma cell lines (MCF-7) were procured from the National Centre for Cell Science, Pune, India, and grown in Eagle’s minimal essential medium containing 10% FBS. The cells were maintained at 37°C, with 5% CO 2 , 95% air, and 100% relative humidity. The cell suspension was treated with trypsin-EDTA and diluted with 5% FBS medium to make up a final density of 1×10 5 cells/mL. An aliquot of 100 µL of cell suspension/well was seeded into a 96-well plate, and later, it was incubated at appropriate conditions. To the above cells, 5 mg of plant extracts dissolved in 100 µl of DMSO was made into a series of 200, 100, 50, 25, and 12.5µg/mL concentrations and added to the respective cell samples. After 24 hrs, the cells were treated with various concentrations of plant extract and incubated at 37°C for 48 hrs. The control was maintained without plant extract, and triplicates were made for all the attention [ 15 ]. Statistical analysis for the cytotoxic assay was performed (n = 3), and the data obtained were represented as mean ± SD. 2.6. LC-MS/MS Analysis ESI calibrant Per the manufacturer's instructions, internal calibration was done using ESI Tune mix (Sigma-Aldrich Chemie G Mb HCO. St. Louis, MO) in negative and positive polarities up to a mass deviation of 0.660 ppm. The mass deviation in ppm is calculated using the formula, Error (ppm) = ((Current mass - reference mass)/reference mass) X 10 6 . 2.6.1. Sample preparation and LC-MS/MS analysis Fraction 1 of CCG was weighed and dissolved in ethyl acetate and chloroform. Screening of compounds present in the plant fractions was performed using LC-HR-ESI-MS/MS analysis (UHPLC Dionex C18 RP Acclaim120 Å, 2.1 × 150 mm, 3.0 µm column, USA and MSMS Bruker Q-II TOF)—sample volume: 500 µL F1 of CCG weighed and dissolved in chloroform. 2.6.2. LC condition : UV at 260 nm; Flow rate − 0.2 mL/min. Discontinuous gradient elution was carried out using mobile phases A and B, represented by acetonitrile: water, respectively (MilliQ), and acidified with formic acid (0.1%). MS condition: ESI, Nebulizer-30.5 psi with 6.0 l/min N2flow, m/z range: 50-1500m/z, Capillary voltage − 4500 V, dry heater temperature at 280 o C, Collision energy − 10eV, Focusing potential − 350 Vpp (Voltage per peak), Transfer time − 80 µS, Prepulse storage − 5µS. MS data were obtained in negative ionization mode [ 16 ]. 2.7. Acridine Orange / Ethidium Bromide Assay (AO/EtBr) MCF-7 cells, grown with specified conditions, were treated with various concentrations of F3 of EAOS and F1 of CCG for 72h at 37°C with 5% CO 2 . Approximately 1µL of a dye mixture (AO = 100 mg/mL and EtBr = 100 mg/mL in distilled water) was assorted by 9 µL cell suspension − 1×10 5 cells/mL on microscope coverslips. The MCF-7 cells were collected, washed using PBS at pH 7.2, and stained with 10 µL of AO and EtBr for 2 min. The cells were cleaned with PBS twice and viewed under a microscope (Nikon Eclipse, Inc, Japan) at a magnification power of 400X, along with an excitation filter at 480 nm [ 17 ]. 2.8. Gene expression studies by semi-quantitative NCL bax, bcl-2 CYCS, and β-actin expression in F1 of CCG-treated MCF-7 cells was determined by semi-quantitative RT-PCR [ 18 ]. Total RNA from MCF-7 cells (1 x 10 5 cells/group) treated with no (control) or with different concentrations (10, 20, 40 µg/mL) of the fraction was isolated after 24 hours. Total RNA was reverse transcribed with primers randomly, oligo (dT) (Quiagen), or a gene-specific primer (GSP) (Quiagen; Custom Plus TaqMan™) using a reverse transcriptase as per the instruction of manufacturers. RT-PCR was performed in a total volume of 20µL. The reaction mixture content is as follows: forward and reverse primer each 0.5µL concentration, 10x master mix (25 mM MgCl2, 10 mM dNTPs, Taq polymerase 2.5 U) 10 µL, cDNA as template 2 µL and remaining volume is nuclease-free dH 2 O 7 µL Amplification cycles were as follows: denatured at 94ºC for 1 minute, cooled at 55ºC for 40 seconds, and extended at 72ºC for 1 minute, for a total of 32 cycles followed by an ending extension at 72ºC for 10 minutes and 16 ºC for 5 min. Finally, the PCR products obtained were subjected to electrophoresis on 1% agarose-TAE gel. Bio-Rad Gel Doc XR scanned the EtBr-stained gels, and the intensity of the product was measured with the help of Image Lab Software version 5 - Bio-Rad. The intensity of the NCL bax, bcl-2, and CYCS band was expressed relative to β- actin [ 18 ]. 2.9. Retrieval of protein and ligands from the database All the protein-small molecule visualization is done using Discovery Studio [ 19 ]. The Ligplots are also generated using Discovery Studio. The docking was performed using the MTi Auto Dock web server (Blind Docking), which is applied here [ 20 ]. The small molecules are retrieved from the PubChem database [ 21 ] in SDF formats, and 3D structures of all the macromolecules are retrieved from the RCSB PDB database. bcl-2, Bax and nucleolin sequences of humans are retrieved from the UniProt database [ 22 ]. The sequences are further subjected to standard protein BLASTP against the PDB database to get the High Query coverage protein structure and Sequence identity. After performing BLAST P [ 23 ], the structure obtained for bcl-2 is PDB-ID 2XA0, which is solved using the X-ray crystallography technique, whereas the Bax protein PDB ID is 1F16 and nucleolin structure has 2KRR as its PDB ID. Results 3.1. In vitro cytotoxic effect of fractions This study utilized an MTT assay to evaluate the cytotoxic effects of the chloroform fraction of Cleome gynandra (CCG) on MCF-7 cells (see Fig. 2 ). The cytotoxic properties of various extracts of C. gynandra are well-documented. The IC 50 values for the CCG fractions (F1-F3) were 82.78, 93.69, and 200 µg/mL, respectively. Of these three fractions, F1 was found to have an excellent cytotoxic effect against MCF-7. The results of the cytotoxic assay showed that the F1 of CCG exhibited an appreciable cytotoxic effect against MCF-7 cell lines. Our findings are consistent with a previous study that observed a dose-dependent anticancer effect of the ethanolic extract of Cleome viscosa on Vero, PC3, HeLa, and MCF-7 cells, as well as the methanolic extract of Cleome gynandra on EAC cells [ 24 , 25 ]. 3.2. Qualitative LC-ESI-MS and LC-ESI-MS/MS F1 of CCG was subjected to LC-MS/MS analysis in a UHPLC system interfaced with ESI-Q-II TOF. The MS/MS fragment peaks were compared with an online mass spectra library like the mass bank to identify the phytocompounds. The LC-MS/MS analysis of F1 of CCG revealed five compounds, namely Tricin (methylated flavone), Pinocembrin (flavone), 4- hydroxy-2,3,4,6 - tetra methoxy chalcone (aromatic ketone), Acacetin (methylated flavone) and Cinnamic acid (Table 1 and Figs. 3 , 4 ). Similar phyto components in the Cleome genus and C.gynandra have been reported in previous studies [ 25 , 26 , 27 ]. The Total Ion Chromatogram (TIC) profile of CCG fractions is shown in Fig. 3 . Acacetin − 5,7-dihydroxy-4'-methoxy flavone compound with anti-plasmodial, anti-inflammatory, anti-mutagenic, anti-oxidant, and anticancer effects rewrite please The Total Ion Chromatogram (TIC) profile of CCG fractions is depicted in Fig. 3 . Acacetin, a 5,7-dihydroxy-4'-methoxy flavone, exhibits various pharmacological activities, including anti-plasmodial, anti-inflammatory, anti-mutagenic, antioxidant, and anticancer effects. Previous studies stated that results of MTT assays denoted that cell viability decreases in a dose-dependent and time-dependent manner in response to Acacetin [ 28 ]. Researchers explored the effects of Acacetin, both in vitro and in vivo , on apoptosis in chronic lymphocytic leukemia (CLL). Additionally, a bio-guided analysis of extracts from Saccharum officinarum led to the isolation of the flavone Tricin-7-O-(6"-methoxycinnamic)-glycoside, known for its notable anti-proliferative and antioxidant properties. [ 29 ]. A researcher reported that in vitro action of flavone Tricin, which is 4',5,7-trihydroxy-3',5', has antiangiogenic solid activity [ 30 ]. Tricin significantly reduced human umbilical vein endothelial cells (HUVECs) at subtoxic levels concerning proliferation, VEGF-induced invasion, and tube formation. Additionally, without exhibiting any cytotoxicity, tricin substantially reduced the angiogenesis of the chorioallantoic membrane in developing chick embryos. One of the principal flavonoids, Pinocembrin (5,7-dihydroxyflavanone), is isolated from various plants. Pinocembrin is an essential flavonoid widely used in the pharmaceutical industry due to its various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidant, and anticancer effects. It has shown anticancer properties against different cancer cells [ 31 ]. Pinocembrin may prevent human prostate cancer cells from proliferation. Its impact on cell proliferation, cell cycle, and apoptosis was evaluated by MTT assay and flow cytometric analysis. Based on the experiment, researchers speculated that pinocembrin stimulates apoptosis of LNCaP human prostate cancer cells accompanied by S and G2/ M phase cell cycle arrest based on the assay [ 32 ]. Cinnamic acid and its natural analogs have been known to treat cancer for centuries [ 33 , 34 ]. Numerous natural and (semi) synthetic chalcones have revealed anticancer activity as a result of their inhibitory potential against a variety of targets, including aromatase, 17'-hydroxysteroid dehydrogenase, 5'-reductase, MMP-2, proteasome, VEGF, VEGFR-2 kinase, ABCG2/Pgp/ BCRP, JAK/STAT, NF-κB, Wnt, signaling pathways, CDC25B, tubulin, HDAC/Situin-1, cathepsin-K, topoisomerase-II, B-Raf, and mTOR, etc [ 35 ]. Researchers investigated the anticancer potential of a novel quinazolinone-chalcone derivative 2-Methyl-3-(3-((E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl)phenyl)-3,4-dihydro-4-quinazolinone (named as 8b) [ 36 ]. These compounds exhibit the best anticancer action both in vitro and in vivo by stopping the cell cycle at the G2/M phase. Similarly, another study exposed that Tetramethoxychalcone (TMOC), through suppressing STAT3 and c-Src activation, inhibits proliferation, prevents cell cycle progression, and also enhances apoptosis in ovarian cancer cells like A2780 and SKOV3 [ 37 ]. Taken together, the data obtained suggest that C. gynandra is having a promising impact against breast cancer cell lines, and further in-depth research will help to reveal the exact mechanism of action for the identified compounds. Table 1 ESI-MS/MS product ions of fractions from CCG Identified Compounds Rt Time Mass Parent ion [M-H] MS/MS [Product ion] Tricin 24 330 329 331,330,329,328,314,299,17. Pinocembrin 28.8–29.0 256 255 255, 213, 171, 169, 145 4-hydroxy-2,3,4,6-Tetra methoxychalcone 24.2–24.6 344 343 343, 328, 313, 298, 285, 270, 255, 249, 242, 183. Acacetin 30.8–31.0 248 283 283,267,227 Cinnamic acid 34.5 356 355 310, 309 3.3. Acridine Orange / Ethidium Bromide Assay (AO/EtBr) The study hypothesized that the F1 fraction of Cleome gynandra (CCG) might induce oxidative stress and apoptosis in MCF-7 cells. While several researchers have demonstrated anticancer activity in selected plants, more convincing evidence should be given regarding the specific phytoconstituents responsible for this activity. Hence, in the present work, attempts were made to explore the possible mechanism for apoptosis induction by the fractions of the selected plants. It was observed that F1 of CCG contains classes of plant flavonoids, which might have up-regulated the expression of bax and CYCS and down-regulated nucleolin and bcl-2 in MCF-7 cells. The results thus suggest that the CCG might have inhibited cell proliferation via apoptotic induction in MCF-7 cells. To understand the mechanism of cell death induced by F1 of CCG treated and untreated, cells were incubated with Ao/EBr − 1:1 at a final concentration of 100µg/mL. AO is an essential dye that can stain both viable and non-viable cells; it can intercalate into DNA, making it appear bright green (an emission maximum at 526nm), and bind to cytoplasmic RNA, making appearances as it was faintly orange. AO/EtBr fluorescent staining is a well-established method to identify the changes after apoptosis induction. After staining, the stages of apoptosis were identified as follows: viable cells display pale green; in the early apoptotic stage, they indicate dark green; in the late apoptotic stage, the cells appear as bright green, and dead cells appear as red fluorescence (Fig. 5 ). Extensive studies have shown that extracts from medicinal plants or plant-derived compounds induced cancer cell death through apoptosis with lesser side effects [ 38 ]. The cytotoxic effect of F1 of CCG on human breast cancer cells was determined by AO/EtBr dual staining. Significant morphological alterations and apoptotic properties such as cell shrinkage, membrane blebbing, and decreased cell density were observed in studies on plant extracts' effects, indicating that the plant has potential anticancer properties [ 39 ]. The study's data are consistent with findings from earlier research [ 40 ]. The staining results indicate that F1 of CCG caused MCF-7 to undergo apoptosis-mediated cell death. In the biological context, ROS are formed as a byproduct of normal oxidative metabolism and have a role in cell signaling. From the images (Fig. 5 ), control cells (Untreated MCF-7 cells) did not display any critical adverse effect compared to cells treated with fractions. The obtained data underscore the cytotoxic effect of the fraction, primarily through the induction of apoptosis [ 41 ]. 3.4. Gene expression studies by semi-quantitative RT-PCR. The interaction of apoptotic and anti-apoptotic proteins governs the fate of cell death and cell survival. Bax, a pro-apoptotic protein, is crucial in mitochondrial-dependent apoptosis induction. bcl-2 is an anti-apoptotic protein that counteracts Bax's function in stimulating cell death. Overexpression of Bax is found to promote apoptotic cell death and decrease tumor enlargement [ 42 ], and on the other hand, its reduced expression inhibits apoptosis [ 43 ]. It has been observed that dose-dependent promising apoptotic effect for the F1 from CCG on MCF-7 cells. Overexpression of bcl-2 protein has been correlated to the improved stability of bcl-2 mRNA [ 44 ]. AU-rich elements in the 3' UTR region of bcl-2-2mRNA show a significant role in mRNA stability. Nucleolin is conserved among eukaryotes and expressed ubiquitously. It has been reported that nucleolin protein binds with AU-rich elements of bcl-2 mRNA, enhancing its strength. Our results indicated that, in a dose-dependent manner, the expression of nucleolin and bcl-2 decreased while the levels of bax and cytochrome c increased in MCF-7 cells. (Figs. 6 & 7 ). the concentration of chloroform extract of F1 of CCG Bax, a protein that belongs to the bcl-2 family, interacts with the bcl-2 protein and promotes apoptosis. The study's observed increase in bax and cytochrome c levels suggests an elevated bax/bcl-2 ratio. The gene expression data align with the previous findings [ 45 , 46 ]. The absence of bcl-2 protein favors the opening of mitochondrial pores, leading to the liberation of cytochrome c into the cytosol, thus inducing apoptosis [ 47 ]. Besides bcl-2, apoptosis can also be mediated through intermediates of ROS [ 48 ]. ROS has been reported in the down-regulation of bcl-2 [ 49 ] and inducing the release of cytochrome C along with Fas-associated proteins recruitment and finally leading to the stimulation of caspase 3 and apoptosis [ 50 ]. Based on the observed results, it is suggested that F1 from CCG-induced apoptosis in MCF- 7 was triggered by the downregulation of bcl-2 and the up-regulation of bax. Earlier studies have shown that Baicalein, a flavonoid, induced apoptosis in cancer cells. Studies showed that the bax protein causes cell death in cancer cells, whereas the bcl-2 protein suppresses the mechanism of apoptosis. [ 32 ] [ 51 ]. Hence, from the previous study reports, down-regulation of bcl-2 in MCF-7 cells could be mediated through free radicals formation and, in turn, induced apoptosis. In this study, the anticancer action and apoptosis mechanism observed in CCG (F1) are primarily attributed to Tricin, 4-oxy-2,3,4,6-tetramethoxychalcone, Pinocembrin, Acacetin, and Cinnamic acid-O-hexoside. These compounds have been reported as antitumor and antioxidant agents. Decreased expression of bcl-2 and nucleolin with a concomitant increase in bax and cytochrome c could stimulate apoptosis in MCF-7 cells. However, the exact mechanism of the down-regulation of bcl-2 and nucleolin and up-regulation of cytochrome C and Bax by the identified compounds remains to be explored. These experimental findings suggest that the anticancer effect exerted by the chloroform extract of C. gynandra was associated with apoptotic induction. This study employed molecular docking to explore the anticancer drugs against breast cancer. Based on binding and docking energy, these studies identify the lead molecules with anticancer potential against human breast cancer. Identifying suitable bioactive compounds from plants has gained more attention in cancer therapy [ 52 ]. Therefore, an in silico docking study was performed on the phytocompounds identified from the selected plant to assess their interaction with various apoptosis-related proteins, particularly bcl-2 and nucleolin. The defective apoptotic pathway is considered the most crucial cause of the occurrence of cancer [ 53 ]. Further, targeting the anti-apoptotic proteins through bioactive compounds is the development in investigating the possible mechanism of action in treating cancer. In this study, naturally occurring ligands - Acacetin, Cinnamic acid, Chalcones, Pinocembrin, and Tricin were docked with bcl-2, nucleolin, and Bax using Schrodinger software (Figs. 8 , 9 , 10 ). Molecular docking of Acacetin, Cinnamic acid, Chalcones, Pinocembrin, and Tricin with ligand bcl-2, Nucleolin, and Bax have an outcome of binding energy level calculations that suit drug modeling of the ligand. 3.5. Lead anticancer compounds and their specificities Energy values of small molecular compounds on the surface grooves of bax, nucleolin, and bcl-2 have been documented (Table 2 ). Table 2. Energy values of smaller compounds Small molecule complex (BAX) Hydrogen bond Energy in Kcal/Mol Acacetin 3 -7.26 Cinnamic Acid 1 -5.58 Pinocembrin 3 -7.01 Tricin 3 -6.59 Small molecule complex (Nucleolin) Hydrogen bond Energy in Kcal/Mol Acacetin 3 -8.46 Cinnamic Acid 2 -5.98 Pinocembrin 3 -8.56 Tricin 4 -8.81 Small molecule complex (bcl-2) Hydrogen bond Energy in Kcal/Mol Acacetin 3 -6.92 Cinnamic Acid - -5.88 Pinocembrin 3 -7.36 Tricin 4 -6.19 Amazingly, ligand molecules exhibited differential binding preference towards anti-apoptotic and pro-apoptotic proteins. The compounds identified from Cleome gynandra that induce apoptosis in cancer cells are observed in silico studies. Previously reported studies on the anticancer efficacy of phytoconstituents [ 54 ] and in vitro experiment findings showed that CCG possesses potent biological activity against cancer. In the present work, the anti-apoptotic proteins bcl-2 and nucleolin have been selected for docking with the compounds identified in the plant extracts. By applying computational approaches, the aim has been to determine the binding energy and identify the specific amino acids involved in the protein's groove. The Bax protein has its highest binding energy with the Acacetin molecule, with an energy of − 7.26 Kcal/Mol. The nucleolin protein has its highest binding energy with the Tricin molecule, with an energy of -8.81 Kcal/Mol. The remaining molecules also have similar energy rates. Hence, all the small molecules have an excellent binding affinity with nucleolin and thus can act as highly potential inhibitors against nucleolin. The bcl-2 protein has its highest binding energy with Pinocembrin, with a binding energy of -7.36 Kcal/Mol. Hence, these molecules are considered suitable potential inhibitors, which is in excellent agreement with the standard and ideal binding energy. These results will be a significant factor in evaluating lead compounds for further drug discovery. In silico studies on the identified molecules, they docked successfully against bcl-2 and nucleolin, suggesting CCG's possible induction of apoptosis in MCF-7. Several chemotherapeutics are designed to disturb proliferation, cause damage to normal cells, and limit their clinical efficiency. Identified molecules also docked effectively against anti-apoptotic proteins, suggesting that these molecules could act as good apoptotic agents; further in-depth studies could lead to the development of a novel anticancer molecule. Conclusion Cleome gynandra L. has been recognized for its different medicinal properties. Hence, an attempt has been made to assess its anti-proliferative effect against the breast cancer cell (MCF7). Thirty-six fractions obtained through column chromatography were analyzed using TLC and then pooled into three fractions (F1-F3) based on their Rf values. Among these, F1 demonstrated the most excellent effectiveness against MCF-7 cells, with an IC 50 value of 82.78 µg/mL. Furthermore, different concentrations of F1 exhibited apoptotic effects in a concentration-dependent manner, as shown by the AO/EBr assay. This study, for the first time, reported the presence of Tricin (methylated flavone), Pinocembrin (flavone), 4-hydroxy-2,3,4,6 - Tetra methoxychalcone (aromatic ketone), Acacetin (methylated flavone) and Cinnamic acid in C. gynandra leaves. Pinocembrin has been reported to exhibit anti-proliferative activity through cell cycle arrest in the S and G2/M phases. Similarly, Cinnamic acid and Tricin have also been reported to possess anticancer activity. Overexpression of bcl-2 and nucleolin prolongs cancer cell survival by inhibiting apoptosis. The effect of F1 on the expression of bcl-2, nucleolin, bax, and cytochrome C was investigated using semi-quantitative RT-PCR with gene-specific primers. The results showed that the expression levels of nucleolin and bcl-2 decreased while the expression of bax and CYCS increased in a concentration-dependent manner. The phytoconstituents present in C.gynandra extract could have induced apoptosis in MCF7 cells either through the down-regulation of anti-apoptotic genes bcl-2 and nucleolin and upregulation of bax and CYSC or by antioxidant activities of flavonoids and other compounds. Acacetin Tricin, Pinocembrin, and Cinnamic acid affinity with bax, Nucleolin, and bcl-2 protein were determined through an in silico approach. These findings suggest that the CCG showed anticancer potential by activating the intrinsic apoptotic pathway in MCF-7 cells. Declarations Authors Contribution: A (Saravanan Renuka) –Data collection and analysis, Manuscript writing, Plagiarism, Data analysis, and References. B (Sridharan Sriram) - LC-MS analysis and interpretation C (Sivakumar Ramalingam) - Concept, review, editing and formatting. Institutional Review Board statement - Not applicable Informed consent statement - Not applicable Data Availability Statement - The data on plant collection and plant material is available at CARISM, SASTRA Deemed to be University, Thanjavur (Voucher Number – CARISM00177). Acknowledgments: Authors thank the Department of Chemistry and Biosciences, SASTRA Deemed to be University, Srinivasa Ramanujan Centre, Kumbakonam. Conflicts of Interest - The authors declare no conflict of interest. Supplementary Materials: Graphical abstract can be prepared and downloaded at: www.https://app.biorender.com. References Mariyappan, S., Ramalingam, S., Murugan, L. & Saravanan, R. 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molecules\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5755739/v1/75c5da5146133daad0ae297e.png"},{"id":73639983,"identity":"362dcae5-077c-401b-949f-b1f028c592fa","added_by":"auto","created_at":"2025-01-13 07:54:27","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":134958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction pattern of Nucleolin and identified molecules\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5755739/v1/131eee39205a5be7c6206b71.png"},{"id":73639981,"identity":"b0f86819-4132-4444-bfa5-a16a021e81ce","added_by":"auto","created_at":"2025-01-13 07:54:27","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":138849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction pattern of Bax with identified molecules\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-5755739/v1/5cf0ff540e646ebc2da8bbe1.png"},{"id":75299758,"identity":"bcf0b02f-e429-4ef6-904c-8acee4399f61","added_by":"auto","created_at":"2025-02-03 07:24:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3983735,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5755739/v1/56875c3a-4980-49af-825e-7e6f8cff8c54.pdf"},{"id":73639974,"identity":"19faa0cf-096b-40b0-8eff-deb95ba35498","added_by":"auto","created_at":"2025-01-13 07:54:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":187470,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-5755739/v1/aa98099405cbfbff310be4e6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eInduction of apoptosis through downregulation of anti-apoptotic genes bcl-2 and Nucleolin in MCF-7 cells by Cleome gynandra leaf fractions containing Tricin, Pinocembrin, and Acacetin\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBreast cancer is the most widespread type of cancer in women worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is a malignant tumor commonly found among Indian women and a significant cause of mortality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Female breast cancer proportion of new cases is 2.3\u0026nbsp;million among all cancer cases, and 6.9 is the mortality proportion of total mortality or mortality due to cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is a heterogeneous cell-cycle deregulatory disorder whose behavior is determined by molecular characteristics. Despite advances in medical screening, therapeutics, and effective prognosis, cancer still poses a broad threat to human health [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Emerging evidence from clinical and epidemiological studies suggests that genetic, environmental, and other pathophysiological conditions are the primary causative agents for cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Breast cancer is often unpredictable, causing premature mortality due to the inadequate efficacy of existing therapeutics. Usually, it is associated with severe side effects. In the past, breast cancer was widespread in industrialized areas. The disease is spread over a vast geographical region and is present in every economic stratum.\u003c/p\u003e \u003cp\u003eBecause of the molecular variation and complexity underlying breast cancer occurrence, its treatment using chemotherapy and radiotherapy is very complicated and often leads to undesirable side effects. Plants and their extracts have been used to treat almost every disease for centuries, and breast cancer is no exception. Herbal medicines contain many chemical compounds, which include alkaloids, flavonoids, glycosides, saponins, resins, and terpenoids. There are complex herbal polyphenols present in the human diet. These compounds counterattack the tumor cells through apoptotic mechanisms [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany plants and their constituents have been found to have promising anti-cancerous properties against breast cancer cells in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models over the past ten years. Because there aren't enough randomized clinical trials, their therapeutic benefits in the treatment of breast cancer are still disputed. A popular approach for identifying potent anticancer drugs is isolating compounds based on bioactivity. Plants are a new, reliable source of anticancer compounds. Numerous essential anticancer medications are now generated from natural sources, including analogs, etoposide analogs, Camptothecin, Indicine N-oxide, Paclitaxel, Topotecan, Vinblastine, and Vincristine [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eAesculusindica, Garcinia mangostana, Pfaffia paniculata, Sapiumellipticum, Solanum nigrum, Artemisia vulgaris\u003c/em\u003e, and \u003cem\u003eVernonia amygdalina\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] are good examples of valuable medicinal plants in cancer treatments. These contain phytocompounds with naturally occurring antioxidant and anticancer properties and are known to inhibit or kill carcinogenic cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCleomaceae as a source of anticancer plants. Traditional medicines play a significant role in India and are frequently the most readily available and cost-effective therapeutic choice for people. The naturally occurring species \u003cem\u003eCleome gynandra\u003c/em\u003e, which belongs to the Cleomaceae family, is often found in and around the Thanjavur region of Tamil Nadu, India. In many African nations, \u003cem\u003eCleome gynandra\u003c/em\u003e is a popular herb and widely dispersed plant located mainly in tropical and subtropical areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Flowers of cleome can be white or yellow. In many places, this herb's seeds and leaves are employed in traditional system of medicine [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Leaf sap has curative powers and is an analgesic to relieve pain from migraines, earaches, \u003cem\u003eetc\u003c/em\u003e. The ears, nose, and eyes are stuffed with fresh leaves.\u003c/p\u003e \u003cp\u003eAdditionally, epileptic fits are treated using it. A combination of leaves or roots is used to alleviate a broad range of ailments, especially severe threadworm infection, conjunctivitis, stomachaches, chest pains, and arthritis can be treated using these leaves. Rheumatism, neuralgia, and headaches can be treated by rubbing them on the affected areas or applying them as a poultice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003eCleome gynandra\u003c/em\u003e is frequently used to treat rheumatoid arthritis, malaria, gonorrhea, and diarrhea. It is employed as an anti-helminthic and rubefacient in India. Scientific evidence has been gathered to support its analgesic, anti-inflammatory, anticancer, and antipyretic effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Because evolutionarily related plant species usually exhibit a high degree of similarity in the types of secondary metabolites and biological activities, these plant characteristics make it a suitable target for anticancer screening. This study is dedicated to reporting the potential of some novel herbal compounds, such as Acacetin, Chalcones, Cinnamic acid, Pinocembrin, and Tricin, to prevent and treat breast cancer. The presence of identified compounds, a well-known antioxidant and anticancer agent, has been reported for the first time in \u003cem\u003eCleome gynandra\u003c/em\u003e. Several studies showed the overexpression of nucleolin and bcl2, the anti-apoptotic genes, in different forms of cancer. Nucleolin has been correlated with increased stability of bcl2 mRNA and helps cancer cells avoid apoptosis (44). Hence, an attempt has been made to assess the cytotoxic effect of \u003cem\u003eCleome gynandra\u003c/em\u003e fractions on MCF7 cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eReagents used in the experiments: medium and Fetal Bovine Serum (FBS) from (GIBCO, USA) and silica gel 100\u0026ndash;200 Mesh (for column chromatography) from Fluka Chemie GmbH (Gesellschaft mit beschr\u0026auml;nkter Haftung, Buchs, Switzerland) were purchased. All chemicals used in the studies were of reagent grade unless otherwise stated and procured from India. TRI Reagent (Sigma- Aldrich Inc. USA), primers \u0026amp; master mix (Quiagen). Unless otherwise noted, all the chemicals used in the studies were of reagent grade and procured from India (Sigma India).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2. Experimental setup\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe leaves of \u003cem\u003eCleome gynandra\u003c/em\u003e L. were collected from Thanjavur (Nov 2018), identified and authenticated by Botanist, CARISM (Centre for Advanced Research in Indian System of Medicine), SASTRA Deemed to be University, Thanjavur, and the herbarium was submitted to CARISM. The same was approved by Dr.N.Ravichandran, Botanist, SASTRA Deemed to be University, CARISM. Identification was also further confirmed compared to specimens recognized at Rapinat Herbarium (RHT, 172), St. Joseph\u0026rsquo;s College, Trichy, Tamil Nadu, India. Methods were carried out by relevant guidelines and regulations of CARISM, SASTRA Deemed to be University, Thanjavur (Voucher Number \u0026ndash; CARISM00177).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2.2.1 Preparation of plant extract\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFresh leaves of \u003cem\u003eCleome gynandra\u003c/em\u003e were washed, dried in an oven, and crushed to a coarse powder. About 100 gm of dried sample was macerated with 150 mL of chloroform and kept at room temperature (Overnight) in a shaker. The extract was collected and dried at 40\u0026ordm;C, allowed to lyophilize using a freeze dryer and stored at 4\u0026ordm;C until use. The crude chloroform extract (5gm) was fractioned by column chromatography and further analyzed by identifying molecules using LC-MS/MS (UHPLC Dionex C18 RP Acclaim 120 \u0026Aring;, 2.1 \u0026times; 150 mm, 3.0 \u0026micro;m column, USA and MSMS Bruker Q-II TOF)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2.3. Crude extract fractionation\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTwo grams of \u003cem\u003eC. gynandra\u003c/em\u003e were lyophilized. The sample was dissolved in 5 mL of chloroform and was then allowed to dry with the mixing of silica gel G (60\u0026ndash;100 mesh). A slurry was prepared and then packed to the column (15 cm) with the help of n-hexane (100%), followed by the mixtures of chloroform, ethyl acetate, and ethanol (90:10, 80:20, 70:30, and finally 50:50) of increasing polarity to obtain fractions. About 36 fractions of CCG were eluted with different solvents with increasing polarity. All the collected fractions were subjected to TLC, and R\u003csub\u003ef\u003c/sub\u003e values were calculated. All 36 CCG fractions were pooled into three fractions (F1-F3) based on their R\u003csub\u003ef\u003c/sub\u003e values.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003e2.4. MTT assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA cytotoxic assay on MCF-7 with CCG was performed using the MTT assay [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. MTT is a tetrazolium salt. The enzyme succinate-dehydrogenase reduces tetrazolium into insoluble formazan, purple. The amount of formazan reflects the number of viable cells present.\u003c/p\u003e \u003cp\u003ePercentage of cell inhibition (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{100-\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{p}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{o}\\text{f}\\:\\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\:\\text{a}\\text{b}\\text{s}\\text{o}\\text{r}\\text{p}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\text{o}\\text{f}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e x 100\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.5.\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanticancer activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe human breast adenocarcinoma cell lines (MCF-7) were procured from the National Centre for Cell Science, Pune, India, and grown in Eagle\u0026rsquo;s minimal essential medium containing 10% FBS. The cells were maintained at 37\u0026deg;C, with 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% air, and 100% relative humidity. The cell suspension was treated with trypsin-EDTA and diluted with 5% FBS medium to make up a final density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL. An aliquot of 100 \u0026micro;L of cell suspension/well was seeded into a 96-well plate, and later, it was incubated at appropriate conditions. To the above cells, 5 mg of plant extracts dissolved in 100 \u0026micro;l of DMSO was made into a series of 200, 100, 50, 25, and 12.5\u0026micro;g/mL concentrations and added to the respective cell samples. After 24 hrs, the cells were treated with various concentrations of plant extract and incubated at 37\u0026deg;C for 48 hrs. The control was maintained without plant extract, and triplicates were made for all the attention [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStatistical analysis for the cytotoxic assay was performed (n\u0026thinsp;=\u0026thinsp;3), and the data obtained were represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. LC-MS/MS Analysis\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eESI calibrant\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePer the manufacturer's instructions, internal calibration was done using ESI Tune mix (Sigma-Aldrich Chemie G Mb HCO. St. Louis, MO) in negative and positive polarities up to a mass deviation of 0.660 ppm. The mass deviation in ppm is calculated using the formula,\u003c/p\u003e \u003cp\u003e \u003cb\u003eError (ppm) = ((Current mass - reference mass)/reference mass) X 10\u003c/b\u003e \u003csup\u003e \u003cb\u003e6\u003c/b\u003e \u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e2.6.1. Sample preparation and LC-MS/MS analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFraction 1 of CCG was weighed and dissolved in ethyl acetate and chloroform. Screening of compounds present in the plant fractions was performed using LC-HR-ESI-MS/MS analysis (UHPLC Dionex C18 RP Acclaim120 \u0026Aring;, 2.1 \u0026times; 150 mm, 3.0 \u0026micro;m column, USA and MSMS Bruker Q-II TOF)\u0026mdash;sample volume: 500 \u0026micro;L F1 of CCG weighed and dissolved in chloroform.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6.2. LC condition\u003c/b\u003e: UV at 260 nm; Flow rate \u0026minus;\u0026thinsp;0.2 mL/min. Discontinuous gradient elution was carried out using mobile phases A and B, represented by acetonitrile: water, respectively (MilliQ), and acidified with formic acid (0.1%). MS condition: ESI, Nebulizer-30.5 psi with 6.0 l/min N2flow, m/z range: 50-1500m/z, Capillary voltage \u0026minus;\u0026thinsp;4500 V, dry heater temperature at 280\u003csup\u003eo\u003c/sup\u003eC, Collision energy \u0026minus;\u0026thinsp;10eV, Focusing potential \u0026minus;\u0026thinsp;350 Vpp (Voltage per peak), Transfer time \u0026minus;\u0026thinsp;80 \u0026micro;S, Prepulse storage \u0026minus;\u0026thinsp;5\u0026micro;S. MS data were obtained in negative ionization mode [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Acridine Orange / Ethidium Bromide Assay (AO/EtBr)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMCF-7 cells, grown with specified conditions, were treated with various concentrations of F3 of EAOS and F1 of CCG for 72h at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Approximately 1\u0026micro;L of a dye mixture (AO\u0026thinsp;=\u0026thinsp;100 mg/mL and EtBr\u0026thinsp;=\u0026thinsp;100 mg/mL in distilled water) was assorted by 9 \u0026micro;L cell suspension \u0026minus;\u0026thinsp;1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL on microscope coverslips. The MCF-7 cells were collected, washed using PBS at pH 7.2, and stained with 10 \u0026micro;L of AO and EtBr for 2 min. The cells were cleaned with PBS twice and viewed under a microscope (Nikon Eclipse, Inc, Japan) at a magnification power of 400X, along with an excitation filter at 480 nm [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Gene expression studies by semi-quantitative\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNCL bax, bcl-2 CYCS, and β-actin expression in F1 of CCG-treated MCF-7 cells was determined by semi-quantitative RT-PCR [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Total RNA from MCF-7 cells (1 x 10\u003csup\u003e5\u003c/sup\u003e cells/group) treated with no (control) or with different concentrations (10, 20, 40 \u0026micro;g/mL) of the fraction was isolated after 24 hours. Total RNA was reverse transcribed with primers randomly, oligo (dT) (Quiagen), or a gene-specific primer (GSP) (Quiagen; Custom Plus TaqMan\u0026trade;) using a reverse transcriptase as per the instruction of manufacturers. RT-PCR was performed in a total volume of 20\u0026micro;L. The reaction mixture content is as follows: forward and reverse primer each 0.5\u0026micro;L concentration, 10x master mix (25 mM MgCl2, 10 mM dNTPs, Taq polymerase 2.5 U) 10 \u0026micro;L, cDNA as template 2 \u0026micro;L and remaining volume is nuclease-free dH\u003csub\u003e2\u003c/sub\u003eO 7 \u0026micro;L Amplification cycles were as follows: denatured at 94\u0026ordm;C for 1 minute, cooled at 55\u0026ordm;C for 40 seconds, and extended at 72\u0026ordm;C for 1 minute, for a total of 32 cycles followed by an ending extension at 72\u0026ordm;C for 10 minutes and 16 \u0026ordm;C for 5 min. Finally, the PCR products obtained were subjected to electrophoresis on 1% agarose-TAE gel. Bio-Rad Gel Doc XR scanned the EtBr-stained gels, and the intensity of the product was measured with the help of Image Lab Software version 5 - Bio-Rad. The intensity of the NCL bax, bcl-2, and CYCS band was expressed relative to β- actin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Retrieval of protein and ligands from the database\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll the protein-small molecule visualization is done using Discovery Studio [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The Ligplots are also generated using Discovery Studio. The docking was performed using the MTi Auto Dock web server (Blind Docking), which is applied here [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The small molecules are retrieved from the PubChem database [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] in SDF formats, and 3D structures of all the macromolecules are retrieved from the RCSB PDB database. bcl-2, Bax and nucleolin sequences of humans are retrieved from the UniProt database [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The sequences are further subjected to standard protein BLASTP against the PDB database to get the High Query coverage protein structure and Sequence identity. After performing BLAST P [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the structure obtained for bcl-2 is PDB-ID 2XA0, which is solved using the X-ray crystallography technique, whereas the Bax protein PDB ID is 1F16 and nucleolin structure has 2KRR as its PDB ID.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003e3.1.\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxic effect of fractions\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eThis study utilized an MTT assay to evaluate the cytotoxic effects of the chloroform fraction of Cleome gynandra (CCG) on MCF-7 cells (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The cytotoxic properties of various extracts of \u003cem\u003eC. gynandra\u003c/em\u003e are well-documented.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe IC\u003csub\u003e50\u003c/sub\u003e values for the CCG fractions (F1-F3) were 82.78, 93.69, and 200 \u0026micro;g/mL, respectively. Of these three fractions, F1 was found to have an excellent cytotoxic effect against MCF-7. The results of the cytotoxic assay showed that the F1 of CCG exhibited an appreciable cytotoxic effect against MCF-7 cell lines. Our findings are consistent with a previous study that observed a dose-dependent anticancer effect of the ethanolic extract of \u003cem\u003eCleome viscosa\u003c/em\u003e on Vero, PC3, HeLa, and MCF-7 cells, as well as the methanolic extract of \u003cem\u003eCleome gynandra\u003c/em\u003e on EAC cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Qualitative LC-ESI-MS and LC-ESI-MS/MS\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eF1 of CCG was subjected to LC-MS/MS analysis in a UHPLC system interfaced with ESI-Q-II TOF. The MS/MS fragment peaks were compared with an online mass spectra library like the mass bank to identify the phytocompounds. The LC-MS/MS analysis of F1 of CCG revealed five compounds, namely Tricin (methylated flavone), Pinocembrin (flavone), 4- hydroxy-2,3,4,6 - tetra methoxy chalcone (aromatic ketone), Acacetin (methylated flavone) and Cinnamic acid (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSimilar phyto components in the Cleome genus and \u003cem\u003eC.gynandra\u003c/em\u003e have been reported in previous studies [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The Total Ion Chromatogram (TIC) profile of CCG fractions is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Acacetin \u0026minus;\u0026thinsp;5,7-dihydroxy-4'-methoxy flavone compound with anti-plasmodial, anti-inflammatory, anti-mutagenic, anti-oxidant, and anticancer effects rewrite please\u003c/p\u003e \u003cp\u003eThe Total Ion Chromatogram (TIC) profile of CCG fractions is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Acacetin, a 5,7-dihydroxy-4'-methoxy flavone, exhibits various pharmacological activities, including anti-plasmodial, anti-inflammatory, anti-mutagenic, antioxidant, and anticancer effects. Previous studies stated that results of MTT assays denoted that cell viability decreases in a dose-dependent and time-dependent manner in response to Acacetin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Researchers explored the effects of Acacetin, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, on apoptosis in chronic lymphocytic leukemia (CLL). Additionally, a bio-guided analysis of extracts from \u003cem\u003eSaccharum officinarum\u003c/em\u003e led to the isolation of the flavone Tricin-7-O-(6\"-methoxycinnamic)-glycoside, known for its notable anti-proliferative and antioxidant properties. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A researcher reported that \u003cem\u003ein vitro\u003c/em\u003e action of flavone Tricin, which is 4',5,7-trihydroxy-3',5', has antiangiogenic solid activity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Tricin significantly reduced human umbilical vein endothelial cells (HUVECs) at subtoxic levels concerning proliferation, VEGF-induced invasion, and tube formation. Additionally, without exhibiting any cytotoxicity, tricin substantially reduced the angiogenesis of the chorioallantoic membrane in developing chick embryos.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eOne of the principal flavonoids, Pinocembrin (5,7-dihydroxyflavanone), is isolated from various plants. Pinocembrin is an essential flavonoid widely used in the pharmaceutical industry due to its various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidant, and anticancer effects. It has shown anticancer properties against different cancer cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Pinocembrin may prevent human prostate cancer cells from proliferation. Its impact on cell proliferation, cell cycle, and apoptosis was evaluated by MTT assay and flow cytometric analysis. Based on the experiment, researchers speculated that pinocembrin stimulates apoptosis of LNCaP human prostate cancer cells accompanied by S and G2/ M phase cell cycle arrest based on the assay [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCinnamic acid and its natural analogs have been known to treat cancer for centuries [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Numerous natural and (semi) synthetic chalcones have revealed anticancer activity as a result of their inhibitory potential against a variety of targets, including aromatase, 17'-hydroxysteroid dehydrogenase, 5'-reductase, MMP-2, proteasome, VEGF, VEGFR-2 kinase, ABCG2/Pgp/ BCRP, JAK/STAT, NF-κB, Wnt, signaling pathways, CDC25B, tubulin, HDAC/Situin-1, cathepsin-K, topoisomerase-II, B-Raf, and mTOR, \u003cem\u003eetc\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eResearchers investigated the anticancer potential of a novel quinazolinone-chalcone derivative 2-Methyl-3-(3-((E)-3-(3,4,5-trimethoxyphenyl)-2-propenyl)phenyl)-3,4-dihydro-4-quinazolinone (named as 8b) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These compounds exhibit the best anticancer action both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by stopping the cell cycle at the G2/M phase. Similarly, another study exposed that Tetramethoxychalcone (TMOC), through suppressing STAT3 and c-Src activation, inhibits proliferation, prevents cell cycle progression, and also enhances apoptosis in ovarian cancer cells like A2780 and SKOV3 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTaken together, the data obtained suggest that \u003cem\u003eC. gynandra\u003c/em\u003e is having a promising impact against breast cancer cell lines, and further in-depth research will help to reveal the exact mechanism of action for the identified compounds.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eESI-MS/MS product ions of fractions from CCG\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIdentified\u003c/p\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRt Time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eParent ion\u003c/p\u003e \u003cp\u003e[M-H]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMS/MS\u003c/p\u003e \u003cp\u003e[Product ion]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTricin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e331,330,329,328,314,299,17.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePinocembrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28.8\u0026ndash;29.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e255, 213, 171, 169, 145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4-hydroxy-2,3,4,6-Tetra methoxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.2\u0026ndash;24.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e344\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e343, 328, 313, 298, 285, 270, 255, 249, 242, 183.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcacetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.8\u0026ndash;31.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e283,267,227\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCinnamic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e310, 309\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Acridine Orange / Ethidium Bromide Assay (AO/EtBr)\u003c/h2\u003e \u003cp\u003eThe study hypothesized that the F1 fraction of \u003cem\u003eCleome gynandra\u003c/em\u003e (CCG) might induce oxidative stress and apoptosis in MCF-7 cells. While several researchers have demonstrated anticancer activity in selected plants, more convincing evidence should be given regarding the specific phytoconstituents responsible for this activity. Hence, in the present work, attempts were made to explore the possible mechanism for apoptosis induction by the fractions of the selected plants. It was observed that F1 of CCG contains classes of plant flavonoids, which might have up-regulated the expression of bax and CYCS and down-regulated nucleolin and bcl-2 in MCF-7 cells. The results thus suggest that the CCG might have inhibited cell proliferation via apoptotic induction in MCF-7 cells.\u003c/p\u003e \u003cp\u003eTo understand the mechanism of cell death induced by F1 of CCG treated and untreated, cells were incubated with Ao/EBr \u0026minus;\u0026thinsp;1:1 at a final concentration of 100\u0026micro;g/mL. AO is an essential dye that can stain both viable and non-viable cells; it can intercalate into DNA, making it appear bright green (an emission maximum at 526nm), and bind to cytoplasmic RNA, making appearances as it was faintly orange. AO/EtBr fluorescent staining is a well-established method to identify the changes after apoptosis induction. After staining, the stages of apoptosis were identified as follows: viable cells display pale green; in the early apoptotic stage, they indicate dark green; in the late apoptotic stage, the cells appear as bright green, and dead cells appear as red fluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExtensive studies have shown that extracts from medicinal plants or plant-derived compounds induced cancer cell death through apoptosis with lesser side effects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The cytotoxic effect of F1 of CCG on human breast cancer cells was determined by AO/EtBr dual staining. Significant morphological alterations and apoptotic properties such as cell shrinkage, membrane blebbing, and decreased cell density were observed in studies on plant extracts' effects, indicating that the plant has potential anticancer properties [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The study's data are consistent with findings from earlier research [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The staining results indicate that F1 of CCG caused MCF-7 to undergo apoptosis-mediated cell death. In the biological context, ROS are formed as a byproduct of normal oxidative metabolism and have a role in cell signaling. From the images (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), control cells (Untreated MCF-7 cells) did not display any critical adverse effect compared to cells treated with fractions. The obtained data underscore the cytotoxic effect of the fraction, primarily through the induction of apoptosis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4. Gene expression studies by semi-quantitative RT-PCR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe interaction of apoptotic and anti-apoptotic proteins governs the fate of cell death and cell survival. Bax, a pro-apoptotic protein, is crucial in mitochondrial-dependent apoptosis induction. bcl-2 is an anti-apoptotic protein that counteracts Bax's function in stimulating cell death. Overexpression of Bax is found to promote apoptotic cell death and decrease tumor enlargement [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and on the other hand, its reduced expression inhibits apoptosis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. It has been observed that dose-dependent promising apoptotic effect for the F1 from CCG on MCF-7 cells.\u003c/p\u003e \u003cp\u003eOverexpression of bcl-2 protein has been correlated to the improved stability of bcl-2 mRNA [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. AU-rich elements in the 3' UTR region of bcl-2-2mRNA show a significant role in mRNA stability. Nucleolin is conserved among eukaryotes and expressed ubiquitously. It has been reported that nucleolin protein binds with AU-rich elements of bcl-2 mRNA, enhancing its strength. Our results indicated that, in a dose-dependent manner, the expression of nucleolin and bcl-2 decreased while the levels of bax and cytochrome c increased in MCF-7 cells. (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u0026amp; \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ethe concentration of chloroform extract of F1 of CCG\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBax, a protein that belongs to the bcl-2 family, interacts with the bcl-2 protein and promotes apoptosis. The study's observed increase in bax and cytochrome c levels suggests an elevated bax/bcl-2 ratio. The gene expression data align with the previous findings [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The absence of bcl-2 protein favors the opening of mitochondrial pores, leading to the liberation of cytochrome c into the cytosol, thus inducing apoptosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Besides bcl-2, apoptosis can also be mediated through intermediates of ROS [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. ROS has been reported in the down-regulation of bcl-2 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and inducing the release of cytochrome C along with Fas-associated proteins recruitment and finally leading to the stimulation of caspase 3 and apoptosis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Based on the observed results, it is suggested that F1 from CCG-induced apoptosis in MCF- 7 was triggered by the downregulation of bcl-2 and the up-regulation of bax.\u003c/p\u003e \u003cp\u003eEarlier studies have shown that Baicalein, a flavonoid, induced apoptosis in cancer cells. Studies showed that the bax protein causes cell death in cancer cells, whereas the bcl-2 protein suppresses the mechanism of apoptosis. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Hence, from the previous study reports, down-regulation of bcl-2 in MCF-7 cells could be mediated through free radicals formation and, in turn, induced apoptosis. In this study, the anticancer action and apoptosis mechanism observed in CCG (F1) are primarily attributed to Tricin, 4-oxy-2,3,4,6-tetramethoxychalcone, Pinocembrin, Acacetin, and Cinnamic acid-O-hexoside. These compounds have been reported as antitumor and antioxidant agents. Decreased expression of bcl-2 and nucleolin with a concomitant increase in bax and cytochrome c could stimulate apoptosis in MCF-7 cells. However, the exact mechanism of the down-regulation of bcl-2 and nucleolin and up-regulation of cytochrome C and Bax by the identified compounds remains to be explored. These experimental findings suggest that the anticancer effect exerted by the chloroform extract of \u003cem\u003eC. gynandra\u003c/em\u003e was associated with apoptotic induction.\u003c/p\u003e \u003cp\u003eThis study employed molecular docking to explore the anticancer drugs against breast cancer. Based on binding and docking energy, these studies identify the lead molecules with anticancer potential against human breast cancer. Identifying suitable bioactive compounds from plants has gained more attention in cancer therapy [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, an \u003cem\u003ein silico\u003c/em\u003e docking study was performed on the phytocompounds identified from the selected plant to assess their interaction with various apoptosis-related proteins, particularly bcl-2 and nucleolin. The defective apoptotic pathway is considered the most crucial cause of the occurrence of cancer [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther, targeting the anti-apoptotic proteins through bioactive compounds is the development in investigating the possible mechanism of action in treating cancer. In this study, naturally occurring ligands - Acacetin, Cinnamic acid, Chalcones, Pinocembrin, and Tricin were docked with bcl-2, nucleolin, and Bax using Schrodinger software (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). Molecular docking of Acacetin, Cinnamic acid, Chalcones, Pinocembrin, and Tricin with ligand bcl-2, Nucleolin, and Bax have an outcome of binding energy level calculations that suit drug modeling of the ligand.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Lead anticancer compounds and their specificities\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEnergy values of small molecular compounds on the surface grooves of bax, nucleolin, and bcl-2 have been documented (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp style='margin-top:3.0pt;margin-right:0in;margin-bottom:3.0pt;margin-left:0in;line-height:normal;font-size:13px;font-family:\"Palatino Linotype\",serif;color:black;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eTable 2. Energy values of smaller compounds\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"border-collapse: collapse;border: none;width: 435px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eSmall molecule complex (BAX)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eHydrogen\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ebond\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.4pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eEnergy in\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eKcal/Mol\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003eAcacetin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003e-7.26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCinnamic Acid\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-5.58\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ePinocembrin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-7.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eTricin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-6.59\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cdiv style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"border-collapse: collapse;border: none;width: 445px;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127.35pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eSmall molecule\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ecomplex (Nucleolin)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.1pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eHydrogen\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ebond\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eEnergy in\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eKcal/Mol\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127.35pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eAcacetin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-8.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127.35pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCinnamic Acid\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-5.98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127.35pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ePinocembrin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92.1pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-8.56\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127.35pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp 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\u003ctr\u003e\n \u003ctd style=\"width: 37.7529%; border-top: 1pt solid windowtext; border-left: none; border-bottom: 1pt solid windowtext; border-right: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eSmall molecule complex (bcl-2)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.5392%; border-top: 1pt solid windowtext; border-left: none; border-bottom: 1pt solid windowtext; border-right: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eHydrogen\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003ebond\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eEnergy in\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eKcal/Mol\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37.7529%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eAcacetin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.5392%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-6.92\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37.7529%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eCinnamic Acid\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.5392%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-5.88\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37.7529%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003ePinocembrin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.5392%; border: none; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;color:red;'\u003e-7.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 37.7529%; border-top: none; border-right: none; border-left: none; border-image: initial; border-bottom: 1pt solid windowtext; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003eTricin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.5392%; border-top: none; border-right: none; border-left: none; border-image: initial; border-bottom: 1pt solid windowtext; padding: 0in 5.4pt; vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113.65pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;font-size:11.0pt;font-family:\"Calibri\",sans-serif;text-align:center;line-height:normal;'\u003e\u003cspan style='font-size:16px;font-family:\"Times New Roman\",serif;'\u003e-6.19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e \u003cp\u003eAmazingly, ligand molecules exhibited differential binding preference towards anti-apoptotic and pro-apoptotic proteins. The compounds identified from \u003cem\u003eCleome gynandra\u003c/em\u003e that induce apoptosis in cancer cells are observed \u003cem\u003ein silico\u003c/em\u003e studies. Previously reported studies on the anticancer efficacy of phytoconstituents [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] and \u003cem\u003ein vitro\u003c/em\u003e experiment findings showed that CCG possesses potent biological activity against cancer.\u003c/p\u003e \u003cp\u003eIn the present work, the anti-apoptotic proteins bcl-2 and nucleolin have been selected for docking with the compounds identified in the plant extracts. By applying computational approaches, the aim has been to determine the binding energy and identify the specific amino acids involved in the protein's groove. The Bax protein has its highest binding energy with the Acacetin molecule, with an energy of \u0026minus;\u0026thinsp;7.26 Kcal/Mol. The nucleolin protein has its highest binding energy with the Tricin molecule, with an energy of -8.81 Kcal/Mol. The remaining molecules also have similar energy rates. Hence, all the small molecules have an excellent binding affinity with nucleolin and thus can act as highly potential inhibitors against nucleolin. The bcl-2 protein has its highest binding energy with Pinocembrin, with a binding energy of -7.36 Kcal/Mol.\u003c/p\u003e \u003cp\u003eHence, these molecules are considered suitable potential inhibitors, which is in excellent agreement with the standard and ideal binding energy. These results will be a significant factor in evaluating lead compounds for further drug discovery. In silico studies on the identified molecules, they docked successfully against bcl-2 and nucleolin, suggesting CCG's possible induction of apoptosis in MCF-7. Several chemotherapeutics are designed to disturb proliferation, cause damage to normal cells, and limit their clinical efficiency. Identified molecules also docked effectively against anti-apoptotic proteins, suggesting that these molecules could act as good apoptotic agents; further in-depth studies could lead to the development of a novel anticancer molecule.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eCleome gynandra\u003c/em\u003e L. has been recognized for its different medicinal properties. Hence, an attempt has been made to assess its anti-proliferative effect against the breast cancer cell (MCF7). Thirty-six fractions obtained through column chromatography were analyzed using TLC and then pooled into three fractions (F1-F3) based on their Rf values. Among these, F1 demonstrated the most excellent effectiveness against MCF-7 cells, with an IC\u003csub\u003e50\u003c/sub\u003e value of 82.78 \u0026micro;g/mL. Furthermore, different concentrations of F1 exhibited apoptotic effects in a concentration-dependent manner, as shown by the AO/EBr assay. This study, for the first time, reported the presence of Tricin (methylated flavone), Pinocembrin (flavone), 4-hydroxy-2,3,4,6 - Tetra methoxychalcone (aromatic ketone), Acacetin (methylated flavone) and Cinnamic acid in \u003cem\u003eC. gynandra\u003c/em\u003e leaves. Pinocembrin has been reported to exhibit anti-proliferative activity through cell cycle arrest in the S and G2/M phases.\u003c/p\u003e \u003cp\u003eSimilarly, Cinnamic acid and Tricin have also been reported to possess anticancer activity. Overexpression of bcl-2 and nucleolin prolongs cancer cell survival by inhibiting apoptosis. The effect of F1 on the expression of bcl-2, nucleolin, bax, and cytochrome C was investigated using semi-quantitative RT-PCR with gene-specific primers. The results showed that the expression levels of nucleolin and bcl-2 decreased while the expression of bax and CYCS increased in a concentration-dependent manner. The phytoconstituents present in \u003cem\u003eC.gynandra\u003c/em\u003e extract could have induced apoptosis in MCF7 cells either through the down-regulation of anti-apoptotic genes bcl-2 and nucleolin and upregulation of bax and CYSC or by antioxidant activities of flavonoids and other compounds. Acacetin Tricin, Pinocembrin, and Cinnamic acid affinity with bax, Nucleolin, and bcl-2 protein were determined through an \u003cem\u003ein silico\u003c/em\u003e approach. These findings suggest that the CCG showed anticancer potential by activating the intrinsic apoptotic pathway in MCF-7 cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors Contribution:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA (Saravanan Renuka) \u0026ndash;Data collection and analysis, Manuscript writing, Plagiarism, Data analysis, and References.\u003c/p\u003e\n\u003cp\u003eB (Sridharan Sriram) - LC-MS analysis and interpretation\u003c/p\u003e\n\u003cp\u003eC (Sivakumar Ramalingam) - Concept, review, editing and formatting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board statement\u003c/strong\u003e - Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent statement\u003c/strong\u003e - Not applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e -\u0026nbsp;The data on plant collection and plant material is available at \u0026nbsp;CARISM, SASTRA Deemed to be University, Thanjavur (Voucher Number \u0026ndash; CARISM00177).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e Authors thank the Department of Chemistry and Biosciences, SASTRA Deemed to be University, Srinivasa Ramanujan Centre, Kumbakonam.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e - The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u003c/strong\u003e Graphical abstract can be prepared and downloaded at:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ewww.https://app.biorender.com.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMariyappan, S., Ramalingam, S., Murugan, L. \u0026amp; Saravanan, R. 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Biochem.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 1813\u0026ndash;1820 (2000).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cleome gynandra, MCF-7, Column chromatography, Apoptosis, Nucleolin and bcl-2","lastPublishedDoi":"10.21203/rs.3.rs-5755739/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5755739/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the anti-proliferative effect of \u003cem\u003eCleome gynandra\u003c/em\u003e leaf fractions on MCF 7 cells. Chloroform extract of \u003cem\u003eCleome gynandra\u003c/em\u003e leaves (CCG) was subjected to column chromatographic separation with increasing polarity of solvents. The total fractions (36) collected were subjected to TLC analysis and pooled into three fractions (F1-F3) based on their R\u003csub\u003ef\u003c/sub\u003e value. Further, these fractions were screened for anti-proliferative and apoptotic effects on MCF7 cells. The IC\u003csub\u003e50\u003c/sub\u003e values for the CCG fractions (F1-F3) were 82.78, 93.69, and 200\u0026micro;g/mL, respectively. Of these fractions, F1 demonstrated a remarkable cytotoxic effect and was subsequently analyzed using LC-MS/MS. For the first time, the plant F1 is found to contain Tricin (a methylated flavone), Pinocembrin (a flavone), 4-hydroxy-2,3,4,6-tetramethoxychalcone (an aromatic ketone), Acacetin (a methylated flavone), and Cinnamic acid. The expression of bax, CYCS, bcl-2, and nucleolin was analyzed using RT-PCR. The MCF 7 cells treated with the F1 showed downregulation of anti-apoptotic genes bcl-2 and nucleolin and upregulation of bax and CYCS. Thus, the induction of apoptosis is mediated through the downregulation of the anti-apoptotic genes.\u003c/p\u003e \u003cp\u003eFurther, an \u003cem\u003ein silico\u003c/em\u003e approach was employed to evaluate the binding affinity of the identified compounds with the Bax, nucleolin, and bcl-2 proteins. It was observed that Acacetin exhibited a stronger binding affinity with bax, Tricin, Nucleolin, and Pinocembrin with bcl-2. Based on this study, Tricin, Pinocembrin, Chalcones, and Cinnamic acid could target the apoptotic and anti-apoptotic genes studied.\u003c/p\u003e","manuscriptTitle":"Induction of apoptosis through downregulation of anti-apoptotic genes bcl-2 and Nucleolin in MCF-7 cells by Cleome gynandra leaf fractions containing Tricin, Pinocembrin, and Acacetin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 07:54:08","doi":"10.21203/rs.3.rs-5755739/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aef28ce3-fd87-4537-b5fb-ead66e4f9e33","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42762998,"name":"Biological sciences/Biochemistry"},{"id":42762999,"name":"Biological sciences/Cancer"}],"tags":[],"updatedAt":"2025-02-03T07:24:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 07:54:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5755739","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5755739","identity":"rs-5755739","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}