Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro

Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro | 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 Research Article Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro Haripriya Shanmugam, R. Narmadha, Caroline Ravikumar, Kiruthika Ariyaperumal, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2951478/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Feb, 2024 Read the published version in Chemical Papers → Version 1 posted 5 You are reading this latest preprint version Abstract Soursop ( Annona muricata ) is being used in treating various types of cancers and there is no report on effect of soursop leaf phytochemicals against osteosarcoma. Current study identified 28 metabolites from ethyl acetate leaf (EAL) extract through GC-MS chemoprofiling and subjected to in silico analysis against the potential protein target, Platelet Derived Growth Factor Receptor α (PDGFRA) of osteosarcoma, including Absorption, Distribution, Metabolism, and Excretion and Toxicity (ADMET) analysis to identify possible hit compounds. This resulted in three hit leaf bioactives namely, 2’- hydroxy-5’-methyl chalcone, linoleic acid and annonacin showing good binding affinity with a docking score of -7.4, -7.0 and – 6.9 kcal/mol respectively. With ADMET analysis, 2’- hydroxy-5’-methyl chalcone and linoleic acid obeyed Lipkinsi’s rule of five, whereas annonacin showed slight violation. Among the three docked complexes, annonacin exhibited good stability during molecular dynamic simulation performed with PDGFRA. Hence, concentration of the key marker compound, annonacin in EAL concentrate is found to be 5.032± 0.13 mg/g of leaf sample. Further, EAL concentrate exhibited cytotoxicity (IC 50 value) on MG-63 osteosarcoma cells in vitro for concentrations ranging from 10 to 25 µg/mL and nuclear imaging of osteoblast cells treated with EAL concentrate at 25 µg/mL concentration exhibited typical symptoms of apoptosis. In vitro cytotoxicity along with nuclear imaging confirmed EAL concentrate from soursop to be a potential drug candidate in developing new anti-cancer agent against osteosarcoma. Soursop leaves Annona muricata GC-MS chemoprofiling Annonacin Osteosarcoma PDGFRA Molecular docking ADMET analysis MG-63 cell lines Nuclear imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction In the process of drug discovery from phytomolecules, synergistic effects of phytochemicals are always preferred over pure compounds in Traditional or Complementary or Integrative systems of Medicine across the globe. However, the key challenge is always to identify the compound(s) responsible for specific bioactivity and toxicity. As a multi-disciplinary approach, the present study focused on designating phytochemicals from leaf extracts of soursop ( Annona muricata) , a plant known for its broad spectrum anti-cancer activity against osteosarcoma or bone cancer through analytical and in silico chemistry approach with confirmation from in vitro cell line studies. Annona muricata L, widely known as graviola or soursop is considered to have potential phytochemicals like phenols, saponins, terpenoids, flavonoids, isoquinoline alkaloids and annonaceous acetogenins (Alali et al. 1999 ; Bonneau et al. 2017 ; Shanmugam et al. 2022 ) that are reported to be effective against various cancer cell lines (Pieme et al. 2014 ). Several research works have confirmed that the entire soursop tree exhibits selective and significant anticancer activity, with leaves being the most potent (Mishra et al. 2013 ; Rupprecht et al. 1990 ). Interestingly, leaf extracts exhibited void/negative impact on normal cells, while showing selective toxicity towards cancerous cells (de Sousa and Vieira, 2010; Oberlies et al. 1995 ). Of late, nano delivery of plant phytochemicals with antiviral properties are explored in cancer treatment to increase the therapeutic efficiency and to overcome the limitations of chemotherapy drugs (Shanmugam et al. 2022 ). Osteosarcoma, a deadly form of musculoskeletal cancer, is the most common type of cancerous tumor in a bone that predominantly affects children and adults (Marques et al. 2014 ; Mora et al. 2019 ). After lymphomas and brain tumors, osteosarcoma is considered to be the third most common cancer occurring in adolescence children with an average diagnosis age of 15 years. Osteosarcoma occurs in primitive mesenchymal bone-forming cells with production of malignant osteoid and exhibits an inclination over the metaphysis of long bones commonly found in the distal femur (43%), proximal tibia (23%) and humerus (10%) (Isakoff et al. 2015 ). Current curative treatment regimens consist of surgery, radiotherapy and intensive chemotherapy. Despite this multi-dimensional approach, survival of osteosarcoma patients is 5 years with 60–65% of survival rate (Blumenthal et al. 2002 ). Even adjuvant chemotherapies results in more side effects with dose intensification and patients are also at high risk of eventual relapse. Among various chemotherapeutic drugs given to the patients, use of high-dose methotrexate, doxorubicin, cisplatin, ifosfamide /etoposide, rafenib, sorafenib and regorafenib have been reported to be effective (Ferguson and Goorin, 2001 ; Rathore and Van Tine, 2021 a). However, these drugs have different targets proteins that inhibit osteosarcoma cell proliferation. Among the various target proteins, receptor tyrosine kinases (RTK) are considered to be the effective target protein inhibiting osteosarcoma proliferation (Ségaliny et al. 2015 ). RTK inhibitors like, Sorafenib and Regorafenib, binds to one of the target receptor, platelet derived growth factor receptors α and β (PDGFR) that has high affinity for various vital polypeptide growth factors, cytokines, hormones and hence, serves as an attractive drug discovery target for osteosarcoma (Zhou et al. 2021 ). Despite the effective inhibition of osteosarcoma proliferations, the current drugs have very high side effects like nerve damage, cardiotoxicity, damage to lining of bladder, renal and liver failure, damage to the white matter of the brain (Bielack et al. 2015 ; Rathore and Van Tine, 2021 b). Considering the side effects, relapse and the cost associated with these treatments, there is a need to identify phytomolcules having selective inhibitory effect on the affected cancerous cells against osteosarcoma. An approach to identify phytomolecules possessing dual or multiple bioactivities like osteogenic stimulatory activity in bael bioactives (Shanmugam et al. 2019 ), apart from anti-cancer activity against osteosarcoma will be interesting. However, the current study is to assess the efficiency of leaf phytochemicals of A. muricata against osteosarcoma and to get an understanding of the chemistry behind the bioactivity. Specifically to identify hits from leaf phytochemicals as potential inhibitors against osteosarcoma targeting the receptor binding protein, Platelet Derived Growth Factor Receptor (PDGFR) through in silico analysis. Various types of PDGFR inhibitors like 3,3′Diindolylmethane, Brazilin, Curcumin, Dehydrozingerone, Ellagic acid, Glyceollins, Pterostilbene, and Vitisin B have been reported from various plants and vegetables sources (Ricci and Ferri, 2015 ). Till date, to the best of our knowledge, there are no reports on the inhibitory effect of A. muricata leaf phytochemicals as potential drug candidate against bone cancer. Hence, assessing the efficiency of leaf phytomolecules of A. muricata against human osteosarcoma and in silico screening of compounds to understand their chemistry could be a potential significant approach in dealing with developing new drug molecules from phytoextracts. To the best of our knowledge, this is the first report on cell proliferation inhibitory effect of leaf phytochemicals of A. muricata against osteosarcoma MG-63 cell lines. 2. Materials and Methods 2.1. Preparation of leaf extract Leaves of A. muricata were collected freshly from the Horticultural Research Station in October 2020, Tamil Nadu Agricultural University, Pechiparai (latitude of 8.44340 N and longitude of 77.30660 E) and transported at room temperature to CANT, TNAU, Coimbatore on the same day. The fresh leaves were graded and shade dried at ambient room temperature of 25 ± 2 ℃ for 5 days, until it reaches constant weight. Dried leaves were extracted with ethyl acetate (AR grade) in the ratio of 1:4 (w/v of leaf powder/solvent) and filtered through filter paper. The filtrates were then concentrated using rotary vacuum evaporator at 45℃ (Heidolph Model-G3, Germany). The obtained leaf concentrates were subjected for further analysis. 2.1.1. GC-MS profiling Ethyl acetate leaf (EAL) concentrate of A. muricata was dissolved in GC-MS grade ethyl acetate and filtered through microfiltration (0.22micron size) technique. The GC-MS analysis was performed on a Perkin Elmer Clarus SQ8C (Perkin Elmer Clarus SQ8C, CA), interfaced to a mass spectrometer (GC-MS) equipped with fused silica column packed with HP-5MS capillary column (TG-5MS, 30 M in length, 0.5mm Diameter, and 0.25 µm film thicknesses). The helium carrier gas was set to 1mL/min flow rate (constant flow mode). The injector temperature was set at 280 0 C. The oven temperature program was programmed to start at 40°C hold for 5 min then ramp to 40–70°C for 2 min, 70°C for 2 min, 70–120°C at 3°C/min, 120–150°C at 5°C/min, 150–220°C at 10°C/min and then 220°C for 2 min. A 1 µL aliquot of the plant samples were injected onto the instrument with a flow rate of carrier gas 1.0 mL/min. The mass spectrometer operating in electron ionization (EI) mode with transfer line temperature at 280°C; ion-source temperature at 230°C; ionization mode; electron impact at 70eV, scan time of 2.88 sec and scanning range of m/z 29–600. Interpretation on mass-spectrum GC-MS was carried out using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. Identification of unknown phytocompounds were carried out by matching the recorded spectra with the data bank mass spectra of NIST library provided with the instrument in- built software. 2.1.2. Quantification of Annonacin The leaf concentrate (1 mg) dissolved in HPLC grade methanol (1 mL), and filtered through a 0.45µm membrane filter, was subjected to quantitative analysis with UV-Vis spectrophotometer (SPECORD PLUS, Analytik Jena AG, Germany) at λ max of 214 nm using standard annonacin (98% HPLC purity) purchased from Aobious technologies, United States of America. The working standard solutions of annonacin were prepared with methanol for six different concentrations in the range of 0.5–10.0µg/mL (annonacin) for obtaining Standard calibration curve with a regression coefficient of 0.99. All spectroscopic conditions were performed at room temperature in triplicates. Concentration of annonacin present in the leaf sample of A. muricata is expressed as milligrams of annonacin per gram of leaf concentrate. 2.2. Molecular docking analysis 2.2.1. Selection of receptors Receptors were selected based on their significant function expressed in the pathway of various types of cancers. The receptor selected for our study was PDGFRA, which is a Type III receptor tyrosine kinase (RTK), a potential target for osteosarcoma. The three-dimensional (3D) coordinates of the selected receptor were retrieved from Protein Data Bank (PDBID: 5K5X) database. The target had a resolution of 2.17 Å, theoretical PI of 8.14, aliphatic index of 86.77, instability index of 46.04, GRAAVY value of -0.256 and a half-life period of 30h. 2.2.2. Structure and active site prediction of proteins The Computed Atlas of Surface Topography of proteins (CASTp) 3.0 was used to predict the active sites that are present in the protein structure (Tian et al. 2018 a). Online server was used for identification and measurement of voids in the 3D protein structures (Tian et al. 2018 b). The 3D protein structures were submitted to the server and the binding site residues have been predicted. 2.2.3. Selection of ligands Compounds from EAL concentrates of A. muricata identified through GC-MS analysis were used for molecular docking study. Structures of 28 phytochemicals including annonacin have been retrieved from PubChem (Kim et al. 2019 ) (Table S1 ) and was used for virtual screening against the potential protein target of bone cancer, PDGFRA. Drug-likeliness, rate of ADMET were predicted for all the 28 phytochemicals inclusive of annonacin. In silico drug-likeliness and toxicity prediction for the identified 28 ligands were carried out using Swiss ADME predictor and the compounds screened based on Lipinski rule of five are listed in Table S2. 2.2.4. Virtual screening of phytochemicals Virtual screening of 28 phytochemicals was performed to study the receptor-ligand interactions, which were considered as the basis for structure-based drug discovery. Docking studies were performed using Python Prescription Virtual Screening tool (PyRx 0.8), which is an open-source software containing AutoDock Vina module (Dallakyan and Olson, 2015 ). The protein receptors and the ligand molecules were converted into pdbqt file using AutoDock module of PyRx tool. The active site residues of the protein target have been defined for grid generation. The grid was generated in the dimensions of X: 35.6973Å, Y: 29.4151Å, Z: 26.3306Å. All the 28 compounds including annonacin were subjected for virtual screening against the target protein, PDGFRA. Two dimensional (2D) interactions between the protein-ligand complexes of top three hit compounds were analyzed based on their binding score using Biovia Discovery Studio Visualizer. 2.2.5 Molecular dynamics simulation The docked complexes of the top three hit compounds of A. muricata were subjected to molecular dynamics simulation study using the GROMACS 5.4.1 suite. The topologies of the protein-ligand complex for three hit compounds were generated with GROMACS utilities using Gromcs94 43a1 force field. The protein-ligand complex has been defined within a unit cubic cell box and solvated with water. For adding ions and to neutralize the protein-ligand complex system, genion tool within GROMACS was used. The molecular dynamics simulation was carried out through energy minimization followed by two steps of equilibration. The energy minimization process was performed through the MD engine of GROMACS mdrun. Then, the system was equilibrated in two phases and mdrun was performed for 10 ns. 2.3. Cell culture studies EAL concentrate containing 28 phytochemicals with estimated amount of annonacin were subjected to in vitro studies against MG-63 human osteosarcoma cell lines (gifted by Dr.Santhini Elango, The South India Textile Research Association, and Coimbatore, India). The cultures were maintained in the medium containing Minimum Essential Media (MEM), Fetal Bovine Serum (FBS) (10% v/v), penicillin (100 units/ml), and streptomycin (100 mg/ml). The cells were incubated at 37°C with 5% CO 2 throughout the study. The cells were then detached after the plates reached 70% confluency using trypsin and seeded as required for the experiments. The experiments were not randomized. 2.3.1 Cytotoxicity assay Leaf concentrate of A. muricata obtained with ethyl acetate were subjected to MTT assay using (Rusanov et al. 2017 ) with slight modifications, a colorimetric assay performed to determine cell viability on MG-63 cells of osteosarcoma. The cells were harvested and seeded in 96-well plates at a concentration of 1 × 10 4 cells/well (Himedia, Mumbai, India) and incubated at 5% CO 2, at 37°C for the cells to adhere to the wells. The culture medium was replaced with the fresh medium containing various IC 50 concentrations (5 to 25 µg/mL) of EAL concentrates of A. muricata to determine its dose dependent anticancer property. After 24 h of EAL concentrate treatment, 20 µL of MTT dye (1 mg/mL) (Sigma Aldrich, Bangalore) was loaded to each well. After 4 h of incubation, purple colored formazon crystals were formed under dark conditions. The supernatant was then carefully pipetted out, and 150 µL of dimethyl Sulfoxide (DMSO) was added to each well. The plates were then placed in a microplate shaker for 5 min and the absorbance at 570 nm was recorded using Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, USA). The culture medium containing cells without EAL concentrate was considered as blank. 2.3.2 Nuclear imaging To monitor the cell division and morphology of MG-63 osteoblast cells, the nuclei were stained with Hoechst 33258 dye (Himedia, Mumbai, India.). After 24 h of treatment with EAL concentrate, morphology of the osteoblast cells were analyzed using fluorescence microscopy (TiS Nikon, Japan). The cells were gently washed with phosphate-buffered saline and treated with Carnoy’s fixation solution (1: 3 acetic acid: methanol) for 10 min. After fixation, Hoechst stain was added and incubated for 30 min, followed by washing with sterile deionized water. The samples were then air dried and observed under an inverted phase contrast epi-fluorescence microscope fitted with a filter having a wavelength range of 460–490 nm (Purschke et al. 2010 ). 2.4. Statistical Analysis All the experiments were carried out in triplicates and data were presented as mean ± SD. The mean difference was tested by One-way analysis of variance (ANOVA), in which level of p< 0.05 was considered as significant. 3. RESULTS AND DISCUSSION 3.1. Chemo-profiling and quantification of annonacin According to the in-house standardized protocol, ethyl acetate has been identified as the best solvent for extraction of the major marker annonaceous acetogenins, annonacin (fatty acid derivatives). In the present study, chemoprofiling of EAL concentrate of A. muricata through GC-MS analysis revealed 27 leaf phytochemicals. It shows major peaks representing fatty acid derivatives, sesquiterpenes, phytosterols, carboxylic acids and esters based on the relative abundance of peak area (Fig. 1 ). Amongst them, Linoleic acid, 2-hydroxy 5-methyl chalcone, Humulene, Tetramethylheptadecan-4-ol, Sitosterol, Phytol, Diethyl Phthalate and Eicosatrienoic acid are found to have reported anti-cancer activity. However, the key and predominant annonaceous acetogenin, annonacin responsible for most anticancer activities are not detected through the GC-MS analysis, as the compounds might have degraded due to the set temperature and duration. Interestingly, annonacin has been detected and quantified through spectroscopic technique with the external standard (98% HPLC purity). Concentration of annonacin present is found to be 5.032 ± 0.13 mg/g of leaf concentrate and the 2D structure of annonacin and is represented in Fig. 1 . Hence, altogether 28 phytochemicals detected and identified through studied analytical methods has been taken for in silico screening against the target protein of osteosarcoma, PDGFRA along with ADMET analysis. The other miscellaneous compounds of less abundance are acetic acid derivatives, epoxides and hydrocarbon compounds. 3.2. In silico analysis of leaf phytochemicals against PDGFRA 3.2.1. Active Site Prediction The promising approach of PDGFR inhibition is directed towards specific targeting of transmembrane RTK present in the bone sarcomas. PDGFRA has been identified as the potential target for osteosarcoma, as selective inhibition of PDGFRA may lead to apoptosis of osteosarcoma cells in vitro (Wilson et al. 2018 ).The binding site has been predicted using CASTp server by submitting the 3D structure of PDGFRA in its PDB file format. The active site residues have been determined from the predicted binding site. The target protein, PDGFRA consists of 18 amino acids in its binding site viz., TRP, LEU, SER, GLN, VAL, MET, LYS, THR, GLY, PRO, ILE, ASN, GLU, TYR, CYS, PHE, ALA and ASP. The active site amino acid residues and its corresponding atoms are given in Table S3. 3.2.2. Analysis of docked compounds with best ligand hits against PDGFRA A structure-based screening of docked phytocompounds is a commonly explored tool during early drug discovery phase. In order to understand the efficiency of leaf phytochemicals in A. muricata to have inhibitory activity against osteosarcoma, in silico analysis has been carried out before in vitro study. Among the 28 phytomolecules docked with the target protein, PDGFRA of osteosarcoma, top three hit compounds has been taken up for further investigation based on their binding affinity score with the interacting amino acid residues. The compounds, 2’- hydroxy-5’-methyl chalcone (interacts with active site residue of Lys 833), linoleic acid (interacts with active site residue of ASP 836) and annonacin (interacts with active site residue of Ser 972 and Gln 828) shows good binding affinity against PDGFRA with a docking score of -7.4, -7.0 and − 6.9 kcal/mol respectively. The 2D molecular structure of 2’ hydroxy − 5’- methyl chalcone (flavonoids), linoleic acid (fatty acid derivative) and annonacin (annonaceous acetogenin) are given in (Fig. S1 ). The 3D view of the complex structure of PDGFRA with the top three hit compounds of A. muricata has been visualized using Pymol and are given in (Table. 1). The docking scores are given in (Table. 2) and the docked images of the interacting residues with the three hit compounds are represented as 2D interaction diagram in (Table. 1). Interestingly, the compound annonacin exhibited a good binding energy of -6.9 Kcal/mol with the target protein, PDGFRA domain by forming 2 hydrogen bonds with the active site residues. Linoleic acid (binding energy of -7.0 Kcal/mol) and 2’- hydroxy-5’-methyl chalcone (binding energy of -7.4kcal/mol) showed lowest binding affinity by forming only single hydrogen bond with the active residue site. The interaction between leaf phytochemicals of A. muricata with the target protein, PDFGRA at specific amino acid residues signifies their role in disruption of oncogenic protein reliability. Table 2 Details of binding energy, number of hydrogen bonds and their interacting amino acids for the three hit compounds S. No Compound Binding energy (kcal/mol) Number of hydrogen bonds formed Interacting amino acids 1. 2’ hydroxy − 5’ methyl chalcone -7.4 1 (Lys A :833) 2. 3. Linoleic acid Annonacin -7.0 -6.9 1 2 (ASP A:836) (Ser A:972, Gln A:828) 3.2.3. In silico ADMET prediction and drug likeness score Potential drug candidates should have certain desirable pharmacokinetic properties to be an effective therapeutic agent. Analysis of in silico ADMET (absorption, distribution, metabolism, excretion, and transport) properties in the early phases of drug discovery reduces failures associated with drug toxicity. Analysis of ADMET related pharmacokinetic and physico-chemical parameters for the 28 leaf phytochemicals of A. muricata has been computed. As seen from (Table. S2), suitability of the identified compounds has been evaluated based on human oral absorption parameters and Lipinski’s rule of five, an indication of drug-likeness for the 28 compounds. According to the results, 27 phytochemicals complied with the Lipinski’s rule of five except annonacin. Among these 28 compounds, only three compounds like 2’- hydroxy-5’-methyl chalcone (Flavonoid), linoleic acid (Polyunsaturated fatty acids) and annonacin (marker acetogenins) are found to have good binding affinity with the target protein, PDGFRA. ADMET analysis for the three hits compounds revealed that the compound, 2’- hydroxy-5’-methyl chalcone is in full accordance with Lipinski’s rule of five; Linoleic acid with one violation (MLogOP > 5) and is found to be within the acceptable range, except for its lipophilic nature with moderate aqueous solubility. The number of rotatable bonds for the compound, 2’- hydroxy-5’-methyl chalcone < 10, signifies an acceptable molecular flexibility with good permeability as well as good oral bioavailability. However, annonacin with molecular weight (596.9 g/mol) more than 500 g/mol has exhibited low gastrointestinal absorption and poor aqueous solubility, indicating its poor bioavailability. These three compounds might serve as potential hits in developing new anti-cancer agent against osteosarcoma. 3.2.4. Molecular Dynamics Simulation The best docked complexes for the top 3 hit compounds are chosen for conducting the molecular dynamics simulation study using the GROMACS 5.4.1 suite. The result of the current simulation study includes Root Mean Square Deviation (RMSD) value, Root Mean Square Fluctuation (RMSF) value and radius of gyration (Rg) for analysis. Molecular dynamics simulation study has been performed to evaluate the stability of the docked complexes with three hit compounds. The simulation for 5k5x with three hit compounds, 2’ hydroxy-5’- methyl chalcone, linoleic acid and annonacin were carried out. RMSD is a key parameter to investigate the equilibrium state of molecular dynamics trajectories. From the present results (Fig. 2 ), RMSD of 5k5x-2’hydroxy-5’- methyl chalcone is found to vary for the first 2.5 ns and then attained an equilibrium state. RMSD of 5k5x-linoleic acid is found to vary for the first 4 ns during the simulation and the attained an equilibrium state. RMSD of 5k5x-annonacin is found to vary only between 6.5 to 7 ns to attain an equilibrium state. Considering the RMSD fluctuations, interaction of annonacin with 5K5X is considered to be stable among the three hit compounds. RMSF of backbone atoms are calculated to check the flexibility of backbone structure in the presence of the ligand and a higher value indicates more flexibility as a result of poor interaction. Mostly similar RMSF has been observed (Fig. 2 ) with both, 2’ hydroxy-5’- methyl chalcone and linoleic acid complexes indicating lesser flexibility with better interaction of proteins. While comparing the other two complexes of 2’ hydroxy-5’- methyl chalcone and linoleic acid, annonacin showed a lower RMSF value with lesser flexibility and better interaction. Radius of gyration evaluates how the secondary structure of protein target gets compactly packed into its 3D form. In general, Rg value indicates the compactness of proteins, which in turn reflects its stability. The more it fluctuates, the less stable it is at that point of time. Hence, Rg value plays a significant role during comparative studies and our results indicated that annonacin while interacting with the protein 5k5x did not affect the stability of the target protein. All these results proved stability of the docked complex of 5k5x with annonacin. In silico analysis plays a significant role in identifying promising drug candidates and in the present study, it designates three major compounds that might exhibit inhibitory or anti-proliferative activity, with annonacin being the most desirable against osteosarcoma. 3.3. Cell line studies 3.3.1. Cell viability The results obtained with little support through in silico analysis, in vitro cytotoxicity assay has been carried out. In the present experiment, the cytotoxicity results of the EAL concentrate of A. muricata with annonacin concentration of 5.032 ± 0.13 mg/g of EAL extract on osteosarcoma cells (MG-63) are summarized in Fig. 3 . It shows that EAL concentrate of A. muricata exhibited cytotoxic activity towards MG-63 cells at all studied IC 50 concentrations (5 to 25 µg/mL). The cell viability showed a decreasing trend, when the concentration has been increased further indicating its significant anticancer activity in MG-63 cells. The trend of decreasing cell viability supports the principle that the leaf extracts rich in key marker acetogenins, annonacin might be responsible for exhibiting inhibitory anti-proliferative property in vitro in MG-63 cells. In addition, presence of the flavonoid, 2’- hydroxy-5’-methyl chalcone could also have provided additional effect through its synergistic activity with annonacin. As, the synergistic interaction between flavonoids and annonaceous acetogenins obtained from leaves of A. muricata has been proven to exert maximum therapeutic efficiency against breast cancer cell lines with enhanced absorption and bioavailability (Yang et al. 2015 ), which supports our current finding. Thus, a quantitative increase in EAL concentrate might have increased the concentration of the major anti-cancer bioactives, specifically annonacin, thought to have contributed to apoptosis on osteosarcoma cells in vitro . Ko et al. 2011 reported that annonacin in MCF-7 breast cancer cells induced growth arrest and apoptosis by inhibiting ERα, cyclin D1 and Bcl-2 protein expressions. 3.4. Nuclear morphology of osteoblast cells Nuclear imaging can be used as a non-destructive molecular visualization tool to know the effect or cytotoxicity caused by the phytomolecules at cellular level. Nuclear morphological abnormalities are considered an essential diagnostic feature to distinguish normal cells from malignant cells. Alterations in nuclear shape of cancer cells can affect transcriptional activity of the cell and thereby gene expressions. In the present study, significant morphological alterations have been observed in the cell nuclei of A. muricata EAL concentrate treated osteoblast cells compared to untreated control cells. Hoechst dye stains the nuclei of the cells regardless of their viability (Baxter et al. 2002 ) and enables them to distinguish the changes that has happened in the morphology of the nuclei induced by the EAL concentrate of A. muricata . Morphology of normal osteoblast cells are spindle-elongated with long lamellipodia (Bielack et al. 2015 ). Also, the untreated/control osteoblast cells retained its original shape and parallel orientation. Interestingly, the EAL concentrate of A. muricata treated cells exhibited nuclei condensation as shown in Fig. 4 . An increase in number of cleaved nuclei has also been observed in the cells treated with EAL concentrate above 10 µg/mL concentration. Cells treated with 25 µg/mL concentration of EAL concentrate exhibited typical characteristics of apoptosis such as cell shrinkage and fragmentation, segregated bodies and cell decrement. A decrease in cell viability along with nuclear imaging results confirmed the inhibitory or anti-proliferative effect of EAL concentrate of A. muricata on MG-63 osteosarcoma cells 4. Conclusion In the present study, 28 leaf phytochemicals identified from A. muricata with known concentration of annonacin content obtained through ethyl acetate has been assessed for its efficacy against osteosarcoma. The in-silico molecular docking analysis of 28 leaf phytochemicals from A. muricata resulted in identification of three hit bioactives namely, 2’- hydroxy-5’-methyl chalcone, linoleic acid and annonacin to be effective against the protein target, PDGFRA of osteosarcoma with a good binding affinity. Amongst the three hit compounds, 2’- hydroxy-5’-methyl chalcone satisfied the Lipinski’s rule of five and ADMET prediction, while linoleic acid and annonacin showed some violations against the rule of five due to their poor aqueous solubility. Though annonacin showed low drug likeness among the three hit compounds due to its lipophilicity, interestingly it showed good binding energy with PDGFRA by forming two hydrogen bonds at the active site of the target protein. On comparing the molecular dynamic behavior of the three hit compounds with target protein (5k5x), annonacin is found to be more stable. Likewise, in-vitro cytotoxicity and nuclear imaging with EAL concentrate of A. muricata treated cells proved its inhibitory action on MG-63 osteosarcoma cells at all studied concentrations. Our in vitro results supports the chemistry of in-silico prediction about the effectiveness of leaf phytochemicals of A. muricata against osteosarcoma. Furthermore, our findings will help researchers to hasten the process of purifying and developing the most efficient therapeutic agent against osteosarcoma from the identified hits or the EAL concentrate without side effects. Declarations Funding sources This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. Data Availability Statement The authors declare that data supporting the findings of this study are available within the article inclusive of supplementary information Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent for Publication All authors have their consent to publish the present work. Conflicts of Interest The authors have no conflicts of interest to declare Author’s contribution HS conceptualized and planed the study. HS sourced, extracted and characterized leaf extract of soursop used in this study at CANT, TNAU. CR, KA and JM carried out molecular interaction studies at CPMBB, TNAU. RN and RS carried out in vitro anti-proliferative activity in MG-63 cancer cells at PSG IAS, Coimbatore. HS drafted the final version of the manuscript and all the authors approved the same. 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J periodon 73:1494-506. Bonneau N, Cynober T, Jullian JC, Champy P (2017) 1H qNMR quantification of Annonaceous acetogenins in crude extracts of Annona muricata L. Fruit Pulp. Phytochem Anal 28:251-56. Dallakyan S, Olson AJ (2015) Small-molecule library screening by docking with PyRx, Chemical biology. Springer pp: 243-50. De Sousa OV, Vieira GD, de Jesus RG de Pinho J, Yamamoto CH, Alves MS (2010) Anti nociceptive and anti-inflammatory activities of the ethanol extract of Annona muricata L. leaves in animal models. Int J Mol Sci 11: 2067-2078. Ferguson WS, Goorin AM (2001) Current treatment of osteosarcoma. Clin Cancer investig J 19: 292-315. Isakoff MS, Bielack SS, Meltzer P, Gorlick R (2015) Osteosarcoma: current treatment and a collaborative pathway to success. J clin oncol 33:3029. Kim S, Chen J, Cheng T, Gindulyte A, He J, He S, Li Q, Shoemaker B, Thiessen P (2019) PubChem 2019 update: Improved access to chemical data, 2019. URL: https://pubchem. ncbi. nlm. nih. gov. doi 10. Ko YM, Wu TY, Wu YC, Chang FR, Guh JY, Chuang LY (2011) Annonacin induces cell cycle-dependent growth arrest and apoptosis in estrogen receptor-α-related pathways in MCF-7 cells. J ethnopharmacol. 137(3):1283-90. Marques C, Ferreira JM, Andronescu E, Ficai D, Sonmez M, Ficai A (2014) Multifunctional materials for bone cancer treatment. Int J Nanomedicine 9:2713. Mishra S, Ahmad S, Kumar N, Sharma B (2013) Annona muricata (the cancer killer): a review. Glob J Pharma Res 2:1613-18. Mora DPP, Santiago KB, Conti BJ, de Oliveira Cardoso E, Conte FL, Oliveira LPG, de Assis Golim M, Uribe JFC, Gutiérrez RM (2019) The chemical composition and events related to the cytotoxic effects of propolis on osteosarcoma cells: A comparative assessment of Colombian samples. Phytother Res 33:591-601. Oberlies NH, Jones JL, Corbett TH, Fotopoulos SS, McLaughlin JL (1995) Tumor cell growth inhibition by several Annonaceous acetogenins in an in vitro disk diffusion assay. Cancer Lett 96:55-62. Pieme CA, Kumar SG, Dongmo MS, Moukette BM, Boyoum FF, Ngogang JY, Saxena AK (2014) Antiproliferative activity and induction of apoptosis by Annona muricata (Annonaceae) extract on human cancer cells. . BMC complement med ther 14:1-10. Purschke M, Rubio N, Held KD, Redmond RW (2010) Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem Photobiol Sci 9:1634-39. Rathore R, Van Tine BA (2021) Pathogenesis and current treatment of osteosarcoma: perspectives for future therapies. J Clin Med 10:1182. Ricci C, Ferri N (2015) Naturally occurring PDGF receptor inhibitors with potential anti-atherosclerotic properties. Vascul Pharmacol 70:1-7. Rupprecht JK, Hui YH, McLaughlin JL (1990) Annonaceous acetogenins: a review. J Nat Prod 53:237-78. Rusanov A, Luzgina N, Lisitsa AV (2017) Sodium dodecyl sulfate cytotoxicity towards HaCaT keratinocytes: comparative analysis of methods for evaluation of cell viability. Bull Exp Biol Med 163:284-88. Ségaliny AI, Tellez-Gabriel M, Heymann MF, Heymann D (2015) Receptor tyrosine kinases: Characterisation, mechanism of action and therapeutic interests for bone cancers. J Bone oncol 4:1-12. Shanmugam H, Dharun VN, Biswal BK, Chandran SV, Vairamani M, Selvamurugan N (2019) Osteogenic stimulatory effect of heraclenin purified from bael in mouse mesenchymal stem cells in vitro . Chem Biol Interact 310:108750. Shanmugam H, Priya B, Swetha MS, Semalaiyappan J (2022) Nano Delivery of Antiviral Plant Bioactives as Cancer Therapeutics, Viral and Antiviral Nanomaterials. CRC Press, pp. 307-49. Tian W, Chen C, Lei X, Zhao J, Liang J (2018) CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res 46: W363-W67. Wilson EA, Russu WA, Shallal HM (2018) Preliminary in vitro and in vivo investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor. Bioorg Med Chem Lett 28:1781-84. Yang C, Gundala SR, Mukkavilli R, Vangala S, Reid MD, Aneja R (2015) Synergistic interactions among flavonoids and acetogenins in Graviola ( Annona muricata ) leaves confer protection against prostate cancer. Carcinogenesis 36 pp: 656-665. Zhou X, Liu H, Zhang M, Li C, Li G (2021) Spectrum‐effect relationship between UPLC fingerprints and anti‐lung cancer effect of Panax ginseng. Phytochem Anal 32:339-46. Table 1 Table 1 is available in the Supplementary Files section. Supplementary Files Table1.docx SupplementaryMaterial.doc Cite Share Download PDF Status: Published Journal Publication published 23 Feb, 2024 Read the published version in Chemical Papers → Version 1 posted Reviewers agreed at journal 30 May, 2023 Reviewers invited by journal 29 May, 2023 Editor invited by journal 20 May, 2023 Editor assigned by journal 19 May, 2023 First submitted to journal 18 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2951478","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":204942045,"identity":"aca1b277-da05-45bd-842e-b4642dc8ddb8","order_by":0,"name":"Haripriya Shanmugam","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBACAyA+wMAgAWIzPoALJxCphdngALFaYIBN4gAuZcjAnP3sw8OFeywS+xvYn1V/3GGX2MB++AHDwx24tVj2pBscnvFMInHGAR6zGwfPJCc28KQZMCSeweOwA2kMh3kOSCQ2HOBhu3GwjTmxgSGHgSGxDY+W888gWuYfYH9WcLCtPrGB/w0BLTegtmw4wGDGcLDtcGKDBCFbbgBtmXFAwnjjYR5jibNtx43bJJ4ZHMDvsDTmzwUH6mTnHW9/+KGyrVq2nz/54cOfeLSAADMQOzYwQ3lsDODIJazFnpCiUTAKRsEoGMEAAC7pVs58wWVqAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-0541-4221","institution":"Tamil Nadu Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Haripriya","middleName":"","lastName":"Shanmugam","suffix":""},{"id":204942046,"identity":"f9821f4d-ff11-4a9a-b66d-769463e60fae","order_by":1,"name":"R. Narmadha","email":"","orcid":"","institution":"PSG Institute of Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Narmadha","suffix":""},{"id":204942047,"identity":"22c920f0-624a-4418-a6ec-1f4ddaa57e3a","order_by":2,"name":"Caroline Ravikumar","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Ravikumar","suffix":""},{"id":204942048,"identity":"51c11425-98a1-4c0c-805a-3f5ce4788d25","order_by":3,"name":"Kiruthika Ariyaperumal","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kiruthika","middleName":"","lastName":"Ariyaperumal","suffix":""},{"id":204942049,"identity":"de5b41ad-41f7-4562-823e-032041563685","order_by":4,"name":"R. Selvakumar","email":"","orcid":"","institution":"PSG Institute of Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Selvakumar","suffix":""},{"id":204942050,"identity":"6c7e3b25-bb2d-4b23-885c-4f26c630149e","order_by":5,"name":"Jayakanthan Mannu","email":"","orcid":"","institution":"Tamil Nadu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jayakanthan","middleName":"","lastName":"Mannu","suffix":""}],"badges":[],"createdAt":"2023-05-18 09:40:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2951478/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2951478/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11696-024-03349-x","type":"published","date":"2024-02-23T15:02:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37771686,"identity":"640abc2d-8c3f-438d-8055-98c98d3ca531","added_by":"auto","created_at":"2023-05-31 14:24:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":546587,"visible":true,"origin":"","legend":"\u003cp\u003ea.\u003cstrong\u003e \u003c/strong\u003eGC-MS chromatogram representing the two hit compounds with their retention time, b. 2D structure of annonacin through colorimetric analysis c. 2D structure of 2-hydroxy 5-Methylchalcone and d. 2D structure of linoleic acid identified through GC-MS phytochemical profiling\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/0b771731606fd3908f0a76f6.png"},{"id":37772887,"identity":"7e3d0765-57ec-49f5-be22-67733a4c75db","added_by":"auto","created_at":"2023-05-31 14:32:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":208205,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dynamics simulation of (a) RMSD of the protein (2’ hydroxy-5’- methyl chalcone), (b) RMSF plot of backbone atoms (2’ hydroxy-5’- methyl chalcone), (c) Radius of gyration (Rg) plot (2’ hydroxy-5’- methyl chalcone), (d) RMSD of the protein (Linoleic acid) (e) RMSF plot of backbone atoms (Linoleic acid), (f) Radius of gyration (Rg) plot (Linoleic acid), (g) RMSD of the protein (Annonacin), (h) RMSF plot of backbone atoms (Annonacin) and (i) Radius of gyration (Rg) plot (Annonacin)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/1bd18d8c6fe0a9e00d1fac10.png"},{"id":37771690,"identity":"17fba8c8-7bad-489d-8ffd-16ee41793be4","added_by":"auto","created_at":"2023-05-31 14:24:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116759,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability (%) of osteoblast cells treated with various concentration of EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/0e8e9a48b63635e978df60f0.png"},{"id":37772888,"identity":"fa506095-01a3-419d-99b0-d1929e6b3fd2","added_by":"auto","created_at":"2023-05-31 14:32:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":948939,"visible":true,"origin":"","legend":"\u003cp\u003eHoechst staining of (a) osteoblast cells (control) and osteoblast cells treated with (b) 5 µg/mL (c) 10 µg/mL (d) 15 µg/mL (e) 20 µg/mL and (f) 25 µg/mL concentration of EAL concentrate containing three hit compounds, 2’ hydroxy-5’- methyl chalcone, linoleic acid and annonacin\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/65eadc3ba7a6c802ab018e8f.png"},{"id":51649419,"identity":"65863856-9e9a-4c3b-a5a3-4c4cc3f76f8c","added_by":"auto","created_at":"2024-02-26 15:16:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1856124,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/e97154bb-a975-4436-b4bd-d063f7ddbf2c.pdf"},{"id":37771688,"identity":"f3b95bb9-cfcf-4bdc-b376-3515fe8b0387","added_by":"auto","created_at":"2023-05-31 14:24:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1348415,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/deaf16057a42a036f6c088f5.docx"},{"id":37771689,"identity":"91d21fc6-2e3f-47b6-838f-df965b8d61aa","added_by":"auto","created_at":"2023-05-31 14:24:52","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1067008,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-2951478/v1/ad73e85f22ec71d44158d755.doc"}],"financialInterests":"","formattedTitle":"Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn the process of drug discovery from phytomolecules, synergistic effects of phytochemicals are always preferred over pure compounds in Traditional or Complementary or Integrative systems of Medicine across the globe. However, the key challenge is always to identify the compound(s) responsible for specific bioactivity and toxicity. As a multi-disciplinary approach, the present study focused on designating phytochemicals from leaf extracts of soursop (\u003cem\u003eAnnona muricata)\u003c/em\u003e, a plant known for its broad spectrum anti-cancer activity against osteosarcoma or bone cancer through analytical and \u003cem\u003ein silico\u003c/em\u003e chemistry approach with confirmation from \u003cem\u003ein vitro\u003c/em\u003e cell line studies. \u003cem\u003eAnnona muricata\u003c/em\u003e L, widely known as graviola or soursop is considered to have potential phytochemicals like phenols, saponins, terpenoids, flavonoids, isoquinoline alkaloids and annonaceous acetogenins (Alali et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Bonneau et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Shanmugam et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) that are reported to be effective against various cancer cell lines (Pieme et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Several research works have confirmed that the entire soursop tree exhibits selective and significant anticancer activity, with leaves being the most potent (Mishra et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rupprecht et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Interestingly, leaf extracts exhibited void/negative impact on normal cells, while showing selective toxicity towards cancerous cells (de Sousa and Vieira, 2010; Oberlies et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Of late, nano delivery of plant phytochemicals with antiviral properties are explored in cancer treatment to increase the therapeutic efficiency and to overcome the limitations of chemotherapy drugs (Shanmugam et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOsteosarcoma, a deadly form of musculoskeletal cancer, is the most common type of cancerous tumor in a bone that predominantly affects children and adults (Marques et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mora et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After lymphomas and brain tumors, osteosarcoma is considered to be the third most common cancer occurring in adolescence children with an average diagnosis age of 15 years. Osteosarcoma occurs in primitive mesenchymal bone-forming cells with production of malignant osteoid and exhibits an inclination over the metaphysis of long bones commonly found in the distal femur (43%), proximal tibia (23%) and humerus (10%) (Isakoff et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Current curative treatment regimens consist of surgery, radiotherapy and intensive chemotherapy. Despite this multi-dimensional approach, survival of osteosarcoma patients is 5 years with 60\u0026ndash;65% of survival rate (Blumenthal et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Even adjuvant chemotherapies results in more side effects with dose intensification and patients are also at high risk of eventual relapse. Among various chemotherapeutic drugs given to the patients, use of high-dose methotrexate, doxorubicin, cisplatin, ifosfamide /etoposide, rafenib, sorafenib and regorafenib have been reported to be effective (Ferguson and Goorin, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rathore and Van Tine, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea). However, these drugs have different targets proteins that inhibit osteosarcoma cell proliferation. Among the various target proteins, receptor tyrosine kinases (RTK) are considered to be the effective target protein inhibiting osteosarcoma proliferation (S\u0026eacute;galiny et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRTK inhibitors like, Sorafenib and Regorafenib, binds to one of the target receptor, platelet derived growth factor receptors α and β (PDGFR) that has high affinity for various vital polypeptide growth factors, cytokines, hormones and hence, serves as an attractive drug discovery target for osteosarcoma (Zhou et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite the effective inhibition of osteosarcoma proliferations, the current drugs have very high side effects like nerve damage, cardiotoxicity, damage to lining of bladder, renal and liver failure, damage to the white matter of the brain (Bielack et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rathore and Van Tine, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb). Considering the side effects, relapse and the cost associated with these treatments, there is a need to identify phytomolcules having selective inhibitory effect on the affected cancerous cells against osteosarcoma. An approach to identify phytomolecules possessing dual or multiple bioactivities like osteogenic stimulatory activity in bael bioactives (Shanmugam et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), apart from anti-cancer activity against osteosarcoma will be interesting. However, the current study is to assess the efficiency of leaf phytochemicals of \u003cem\u003eA. muricata\u003c/em\u003e against osteosarcoma and to get an understanding of the chemistry behind the bioactivity. Specifically to identify hits from leaf phytochemicals as potential inhibitors against osteosarcoma targeting the receptor binding protein, Platelet Derived Growth Factor Receptor (PDGFR) through \u003cem\u003ein silico\u003c/em\u003e analysis. Various types of PDGFR inhibitors like 3,3\u0026prime;Diindolylmethane, Brazilin, Curcumin, Dehydrozingerone, Ellagic acid, Glyceollins, Pterostilbene, and Vitisin B have been reported from various plants and vegetables sources (Ricci and Ferri, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Till date, to the best of our knowledge, there are no reports on the inhibitory effect of \u003cem\u003eA. muricata\u003c/em\u003e leaf phytochemicals as potential drug candidate against bone cancer. Hence, assessing the efficiency of leaf phytomolecules of \u003cem\u003eA. muricata\u003c/em\u003e against human osteosarcoma and \u003cem\u003ein silico\u003c/em\u003e screening of compounds to understand their chemistry could be a potential significant approach in dealing with developing new drug molecules from phytoextracts. To the best of our knowledge, this is the first report on cell proliferation inhibitory effect of leaf phytochemicals of \u003cem\u003eA. muricata\u003c/em\u003e against osteosarcoma MG-63 cell lines.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Preparation of leaf extract\u003c/h2\u003e \u003cp\u003eLeaves of \u003cem\u003eA. muricata\u003c/em\u003e were collected freshly from the Horticultural Research Station in October 2020, Tamil Nadu Agricultural University, Pechiparai (latitude of 8.44340 N and longitude of 77.30660 E) and transported at room temperature to CANT, TNAU, Coimbatore on the same day. The fresh leaves were graded and shade dried at ambient room temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 ℃ for 5 days, until it reaches constant weight. Dried leaves were extracted with ethyl acetate (AR grade) in the ratio of 1:4 (w/v of leaf powder/solvent) and filtered through filter paper. The filtrates were then concentrated using rotary vacuum evaporator at 45℃ (Heidolph Model-G3, Germany). The obtained leaf concentrates were subjected for further analysis.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. GC-MS profiling\u003c/h2\u003e \u003cp\u003eEthyl acetate leaf (EAL) concentrate of \u003cem\u003eA. muricata\u003c/em\u003e was dissolved in GC-MS grade ethyl acetate and filtered through microfiltration (0.22micron size) technique. The GC-MS analysis was performed on a Perkin Elmer Clarus SQ8C (Perkin Elmer Clarus SQ8C, CA), interfaced to a mass spectrometer (GC-MS) equipped with fused silica column packed with HP-5MS capillary column (TG-5MS, 30 M in length, 0.5mm Diameter, and 0.25 \u0026micro;m film thicknesses). The helium carrier gas was set to 1mL/min flow rate (constant flow mode). The injector temperature was set at 280\u003csup\u003e0\u003c/sup\u003eC. The oven temperature program was programmed to start at 40\u0026deg;C hold for 5 min then ramp to 40\u0026ndash;70\u0026deg;C for 2 min, 70\u0026deg;C for 2 min, 70\u0026ndash;120\u0026deg;C at 3\u0026deg;C/min, 120\u0026ndash;150\u0026deg;C at 5\u0026deg;C/min, 150\u0026ndash;220\u0026deg;C at 10\u0026deg;C/min and then 220\u0026deg;C for 2 min. A 1 \u0026micro;L aliquot of the plant samples were injected onto the instrument with a flow rate of carrier gas 1.0 mL/min. The mass spectrometer operating in electron ionization (EI) mode with transfer line temperature at 280\u0026deg;C; ion-source temperature at 230\u0026deg;C; ionization mode; electron impact at 70eV, scan time of 2.88 sec and scanning range of m/z 29\u0026ndash;600. Interpretation on mass-spectrum GC-MS was carried out using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. Identification of unknown phytocompounds were carried out by matching the recorded spectra with the data bank mass spectra of NIST library provided with the instrument in- built software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Quantification of Annonacin\u003c/h2\u003e \u003cp\u003eThe leaf concentrate (1 mg) dissolved in HPLC grade methanol (1 mL), and filtered through a 0.45\u0026micro;m membrane filter, was subjected to quantitative analysis with UV-Vis spectrophotometer (SPECORD PLUS, Analytik Jena AG, Germany) at λ\u003csub\u003emax\u003c/sub\u003e of 214 nm using standard annonacin (98% HPLC purity) purchased from Aobious technologies, United States of America. The working standard solutions of annonacin were prepared with methanol for six different concentrations in the range of 0.5\u0026ndash;10.0\u0026micro;g/mL (annonacin) for obtaining Standard calibration curve with a regression coefficient of 0.99. All spectroscopic conditions were performed at room temperature in triplicates. Concentration of annonacin present in the leaf sample of \u003cem\u003eA. muricata\u003c/em\u003e is expressed as milligrams of annonacin per gram of leaf concentrate.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Molecular docking analysis\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Selection of receptors\u003c/h2\u003e \u003cp\u003eReceptors were selected based on their significant function expressed in the pathway of various types of cancers. The receptor selected for our study was PDGFRA, which is a Type III receptor tyrosine kinase (RTK), a potential target for osteosarcoma. The three-dimensional (3D) coordinates of the selected receptor were retrieved from Protein Data Bank (PDBID: 5K5X) database. The target had a resolution of 2.17 \u0026Aring;, theoretical PI of 8.14, aliphatic index of 86.77, instability index of 46.04, GRAAVY value of -0.256 and a half-life period of 30h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. Structure and active site prediction of proteins\u003c/h2\u003e \u003cp\u003eThe Computed Atlas of Surface Topography of proteins (CASTp) 3.0 was used to predict the active sites that are present in the protein structure (Tian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003ea). Online server was used for identification and measurement of voids in the 3D protein structures (Tian et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003eb). The 3D protein structures were submitted to the server and the binding site residues have been predicted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3. Selection of ligands\u003c/h2\u003e \u003cp\u003eCompounds from EAL concentrates of \u003cem\u003eA. muricata\u003c/em\u003e identified through GC-MS analysis were used for molecular docking study. Structures of 28 phytochemicals including annonacin have been retrieved from PubChem (Kim et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and was used for virtual screening against the potential protein target of bone cancer, PDGFRA. Drug-likeliness, rate of ADMET were predicted for all the 28 phytochemicals inclusive of annonacin. \u003cem\u003eIn silico\u003c/em\u003e drug-likeliness and toxicity prediction for the identified 28 ligands were carried out using Swiss ADME predictor and the compounds screened based on Lipinski rule of five are listed in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4. Virtual screening of phytochemicals\u003c/h2\u003e \u003cp\u003eVirtual screening of 28 phytochemicals was performed to study the receptor-ligand interactions, which were considered as the basis for structure-based drug discovery. Docking studies were performed using Python Prescription Virtual Screening tool (PyRx 0.8), which is an open-source software containing AutoDock Vina module (Dallakyan and Olson, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The protein receptors and the ligand molecules were converted into pdbqt file using AutoDock module of PyRx tool. The active site residues of the protein target have been defined for grid generation. The grid was generated in the dimensions of X: 35.6973\u0026Aring;, Y: 29.4151\u0026Aring;, Z: 26.3306\u0026Aring;. All the 28 compounds including annonacin were subjected for virtual screening against the target protein, PDGFRA. Two dimensional (2D) interactions between the protein-ligand complexes of top three hit compounds were analyzed based on their binding score using Biovia Discovery Studio Visualizer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.2.5 Molecular dynamics simulation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe docked complexes of the top three hit compounds of \u003cem\u003eA. muricata\u003c/em\u003e were subjected to molecular dynamics simulation study using the GROMACS 5.4.1 suite. The topologies of the protein-ligand complex for three hit compounds were generated with GROMACS utilities using Gromcs94 43a1 force field. The protein-ligand complex has been defined within a unit cubic cell box and solvated with water. For adding ions and to neutralize the protein-ligand complex system, genion tool within GROMACS was used. The molecular dynamics simulation was carried out through energy minimization followed by two steps of equilibration. The energy minimization process was performed through the MD engine of GROMACS mdrun. Then, the system was equilibrated in two phases and mdrun was performed for 10 ns.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell culture studies\u003c/h2\u003e \u003cp\u003eEAL concentrate containing 28 phytochemicals with estimated amount of annonacin were subjected to \u003cem\u003ein vitro\u003c/em\u003e studies against MG-63 human osteosarcoma cell lines (gifted by Dr.Santhini Elango, The South India Textile Research Association, and Coimbatore, India). The cultures were maintained in the medium containing Minimum Essential Media (MEM), Fetal Bovine Serum (FBS) (10% v/v), penicillin (100 units/ml), and streptomycin (100 mg/ml). The cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e throughout the study. The cells were then detached after the plates reached 70% confluency using trypsin and seeded as required for the experiments. The experiments were not randomized.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Cytotoxicity assay\u003c/h2\u003e \u003cp\u003eLeaf concentrate of \u003cem\u003eA. muricata\u003c/em\u003e obtained with ethyl acetate were subjected to MTT assay using (Rusanov et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) with slight modifications, a colorimetric assay performed to determine cell viability on MG-63 cells of osteosarcoma. The cells were harvested and seeded in 96-well plates at a concentration of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well (Himedia, Mumbai, India) and incubated at 5% CO\u003csub\u003e2,\u003c/sub\u003e at 37\u0026deg;C for the cells to adhere to the wells. The culture medium was replaced with the fresh medium containing various IC\u003csub\u003e50\u003c/sub\u003e concentrations (5 to 25 \u0026micro;g/mL) of EAL concentrates of \u003cem\u003eA. muricata\u003c/em\u003e to determine its dose dependent anticancer property. After 24 h of EAL concentrate treatment, 20 \u0026micro;L of MTT dye (1 mg/mL) (Sigma Aldrich, Bangalore) was loaded to each well. After 4 h of incubation, purple colored formazon crystals were formed under dark conditions. The supernatant was then carefully pipetted out, and 150 \u0026micro;L of dimethyl Sulfoxide (DMSO) was added to each well. The plates were then placed in a microplate shaker for 5 min and the absorbance at 570 nm was recorded using Multiskan\u0026trade; GO Microplate Spectrophotometer (Thermo Scientific, USA). The culture medium containing cells without EAL concentrate was considered as blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Nuclear imaging\u003c/h2\u003e \u003cp\u003eTo monitor the cell division and morphology of MG-63 osteoblast cells, the nuclei were stained with Hoechst 33258 dye (Himedia, Mumbai, India.). After 24 h of treatment with EAL concentrate, morphology of the osteoblast cells were analyzed using fluorescence microscopy (TiS Nikon, Japan). The cells were gently washed with phosphate-buffered saline and treated with Carnoy\u0026rsquo;s fixation solution (1: 3 acetic acid: methanol) for 10 min. After fixation, Hoechst stain was added and incubated for 30 min, followed by washing with sterile deionized water. The samples were then air dried and observed under an inverted phase contrast epi-fluorescence microscope fitted with a filter having a wavelength range of 460\u0026ndash;490 nm (Purschke et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were carried out in triplicates and data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The mean difference was tested by One-way analysis of variance (ANOVA), in which level of p\u0026lt; 0.05 was considered as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Chemo-profiling and quantification of annonacin\u003c/h2\u003e \u003cp\u003eAccording to the in-house standardized protocol, ethyl acetate has been identified as the best solvent for extraction of the major marker annonaceous acetogenins, annonacin (fatty acid derivatives). In the present study, chemoprofiling of EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e through GC-MS analysis revealed 27 leaf phytochemicals. It shows major peaks representing fatty acid derivatives, sesquiterpenes, phytosterols, carboxylic acids and esters based on the relative abundance of peak area (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Amongst them, Linoleic acid, 2-hydroxy 5-methyl chalcone, Humulene, Tetramethylheptadecan-4-ol, Sitosterol, Phytol, Diethyl Phthalate and Eicosatrienoic acid are found to have reported anti-cancer activity. However, the key and predominant annonaceous acetogenin, annonacin responsible for most anticancer activities are not detected through the GC-MS analysis, as the compounds might have degraded due to the set temperature and duration. Interestingly, annonacin has been detected and quantified through spectroscopic technique with the external standard (98% HPLC purity). Concentration of annonacin present is found to be 5.032\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/g of leaf concentrate and the 2D structure of annonacin and is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Hence, altogether 28 phytochemicals detected and identified through studied analytical methods has been taken for \u003cem\u003ein silico\u003c/em\u003e screening against the target protein of osteosarcoma, PDGFRA along with ADMET analysis. The other miscellaneous compounds of less abundance are acetic acid derivatives, epoxides and hydrocarbon compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2. \u003cem\u003eIn silico\u003c/em\u003e analysis of leaf phytochemicals against PDGFRA\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. Active Site Prediction\u003c/h2\u003e \u003cp\u003eThe promising approach of PDGFR inhibition is directed towards specific targeting of transmembrane RTK present in the bone sarcomas. PDGFRA has been identified as the potential target for osteosarcoma, as selective inhibition of PDGFRA may lead to apoptosis of osteosarcoma cells \u003cem\u003ein vitro\u003c/em\u003e (Wilson et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).The binding site has been predicted using CASTp server by submitting the 3D structure of PDGFRA in its PDB file format. The active site residues have been determined from the predicted binding site. The target protein, PDGFRA consists of 18 amino acids in its binding site viz., TRP, LEU, SER, GLN, VAL, MET, LYS, THR, GLY, PRO, ILE, ASN, GLU, TYR, CYS, PHE, ALA and ASP. The active site amino acid residues and its corresponding atoms are given in Table S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. Analysis of docked compounds with best ligand hits against PDGFRA\u003c/h2\u003e \u003cp\u003eA structure-based screening of docked phytocompounds is a commonly explored tool during early drug discovery phase. In order to understand the efficiency of leaf phytochemicals in \u003cem\u003eA. muricata\u003c/em\u003e to have inhibitory activity against osteosarcoma, \u003cem\u003ein silico\u003c/em\u003e analysis has been carried out before \u003cem\u003ein vitro\u003c/em\u003e study. Among the 28 phytomolecules docked with the target protein, PDGFRA of osteosarcoma, top three hit compounds has been taken up for further investigation based on their binding affinity score with the interacting amino acid residues. The compounds, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone (interacts with active site residue of Lys 833), linoleic acid (interacts with active site residue of ASP 836) and annonacin (interacts with active site residue of Ser 972 and Gln 828) shows good binding affinity against PDGFRA with a docking score of -7.4, -7.0 and \u0026minus;\u0026thinsp;6.9 kcal/mol respectively. The 2D molecular structure of 2\u0026rsquo; hydroxy \u0026minus;\u0026thinsp;5\u0026rsquo;- methyl chalcone (flavonoids), linoleic acid (fatty acid derivative) and annonacin (annonaceous acetogenin) are given in (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 3D view of the complex structure of PDGFRA with the top three hit compounds of \u003cem\u003eA. muricata\u003c/em\u003e has been visualized using Pymol and are given in (Table. 1). The docking scores are given in (Table. 2) and the docked images of the interacting residues with the three hit compounds are represented as 2D interaction diagram in (Table. 1). Interestingly, the compound annonacin exhibited a good binding energy of -6.9 Kcal/mol with the target protein, PDGFRA domain by forming 2 hydrogen bonds with the active site residues. Linoleic acid (binding energy of -7.0 Kcal/mol) and 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone (binding energy of -7.4kcal/mol) showed lowest binding affinity by forming only single hydrogen bond with the active residue site. The interaction between leaf phytochemicals of \u003cem\u003eA. muricata\u003c/em\u003e with the target protein, PDFGRA at specific amino acid residues signifies their role in disruption of oncogenic protein reliability.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of binding energy, number of hydrogen bonds and their interacting amino acids for the three hit compounds\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\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBinding energy (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNumber of hydrogen bonds formed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInteracting amino acids\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u0026rsquo; hydroxy \u0026minus;\u0026thinsp;5\u0026rsquo; methyl chalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(Lys A :833)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLinoleic acid\u003c/p\u003e \u003cp\u003eAnnonacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-7.0\u003c/p\u003e \u003cp\u003e-6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(ASP A:836)\u003c/p\u003e \u003cp\u003e(Ser A:972, Gln A:828)\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=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. \u003cem\u003eIn silico\u003c/em\u003e ADMET prediction and drug likeness score\u003c/h2\u003e \u003cp\u003ePotential drug candidates should have certain desirable pharmacokinetic properties to be an effective therapeutic agent. Analysis of \u003cem\u003ein silico\u003c/em\u003e ADMET (absorption, distribution, metabolism, excretion, and transport) properties in the early phases of drug discovery reduces failures associated with drug toxicity. Analysis of ADMET related pharmacokinetic and physico-chemical parameters for the 28 leaf phytochemicals of \u003cem\u003eA. muricata\u003c/em\u003e has been computed. As seen from (Table. S2), suitability of the identified compounds has been evaluated based on human oral absorption parameters and Lipinski\u0026rsquo;s rule of five, an indication of drug-likeness for the 28 compounds. According to the results, 27 phytochemicals complied with the Lipinski\u0026rsquo;s rule of five except annonacin. Among these 28 compounds, only three compounds like 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone (Flavonoid), linoleic acid (Polyunsaturated fatty acids) and annonacin (marker acetogenins) are found to have good binding affinity with the target protein, PDGFRA. ADMET analysis for the three hits compounds revealed that the compound, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone is in full accordance with Lipinski\u0026rsquo;s rule of five; Linoleic acid with one violation (MLogOP\u0026thinsp;\u0026gt;\u0026thinsp;5) and is found to be within the acceptable range, except for its lipophilic nature with moderate aqueous solubility. The number of rotatable bonds for the compound, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone\u0026thinsp;\u0026lt;\u0026thinsp;10, signifies an acceptable molecular flexibility with good permeability as well as good oral bioavailability. However, annonacin with molecular weight (596.9 g/mol) more than 500 g/mol has exhibited low gastrointestinal absorption and poor aqueous solubility, indicating its poor bioavailability. These three compounds might serve as potential hits in developing new anti-cancer agent against osteosarcoma.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. Molecular Dynamics Simulation\u003c/h2\u003e \u003cp\u003eThe best docked complexes for the top 3 hit compounds are chosen for conducting the molecular dynamics simulation study using the GROMACS 5.4.1 suite. The result of the current simulation study includes Root Mean Square Deviation (RMSD) value, Root Mean Square Fluctuation (RMSF) value and radius of gyration (Rg) for analysis. Molecular dynamics simulation study has been performed to evaluate the stability of the docked complexes with three hit compounds. The simulation for 5k5x with three hit compounds, 2\u0026rsquo; hydroxy-5\u0026rsquo;- methyl chalcone, linoleic acid and annonacin were carried out. RMSD is a key parameter to investigate the equilibrium state of molecular dynamics trajectories. From the present results (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), RMSD of 5k5x-2\u0026rsquo;hydroxy-5\u0026rsquo;- methyl chalcone is found to vary for the first 2.5 ns and then attained an equilibrium state. RMSD of 5k5x-linoleic acid is found to vary for the first 4 ns during the simulation and the attained an equilibrium state. RMSD of 5k5x-annonacin is found to vary only between 6.5 to 7 ns to attain an equilibrium state. Considering the RMSD fluctuations, interaction of annonacin with 5K5X is considered to be stable among the three hit compounds. RMSF of backbone atoms are calculated to check the flexibility of backbone structure in the presence of the ligand and a higher value indicates more flexibility as a result of poor interaction. Mostly similar RMSF has been observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with both, 2\u0026rsquo; hydroxy-5\u0026rsquo;- methyl chalcone and linoleic acid complexes indicating lesser flexibility with better interaction of proteins. While comparing the other two complexes of 2\u0026rsquo; hydroxy-5\u0026rsquo;- methyl chalcone and linoleic acid, annonacin showed a lower RMSF value with lesser flexibility and better interaction.\u003c/p\u003e \u003cp\u003eRadius of gyration evaluates how the secondary structure of protein target gets compactly packed into its 3D form. In general, Rg value indicates the compactness of proteins, which in turn reflects its stability. The more it fluctuates, the less stable it is at that point of time. Hence, Rg value plays a significant role during comparative studies and our results indicated that annonacin while interacting with the protein 5k5x did not affect the stability of the target protein. All these results proved stability of the docked complex of 5k5x with annonacin. \u003cem\u003eIn silico\u003c/em\u003e analysis plays a significant role in identifying promising drug candidates and in the present study, it designates three major compounds that might exhibit inhibitory or anti-proliferative activity, with annonacin being the most desirable against osteosarcoma.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cell line studies\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Cell viability\u003c/h2\u003e \u003cp\u003eThe results obtained with little support through \u003cem\u003ein silico\u003c/em\u003e analysis, \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity assay has been carried out. In the present experiment, the cytotoxicity results of the EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e with annonacin concentration of 5.032\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/g of EAL extract on osteosarcoma cells (MG-63) are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It shows that EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e exhibited cytotoxic activity towards MG-63 cells at all studied IC\u003csub\u003e50\u003c/sub\u003e concentrations (5 to 25 \u0026micro;g/mL). The cell viability showed a decreasing trend, when the concentration has been increased further indicating its significant anticancer activity in MG-63 cells. The trend of decreasing cell viability supports the principle that the leaf extracts rich in key marker acetogenins, annonacin might be responsible for exhibiting inhibitory anti-proliferative property \u003cem\u003ein vitro\u003c/em\u003e in MG-63 cells. In addition, presence of the flavonoid, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone could also have provided additional effect through its synergistic activity with annonacin. As, the synergistic interaction between flavonoids and annonaceous acetogenins obtained from leaves of \u003cem\u003eA. muricata\u003c/em\u003e has been proven to exert maximum therapeutic efficiency against breast cancer cell lines with enhanced absorption and bioavailability (Yang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), which supports our current finding. Thus, a quantitative increase in EAL concentrate might have increased the concentration of the major anti-cancer bioactives, specifically annonacin, thought to have contributed to apoptosis on osteosarcoma cells \u003cem\u003ein vitro\u003c/em\u003e. Ko et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e reported that annonacin in MCF-7 breast cancer cells induced growth arrest and apoptosis by inhibiting ERα, cyclin D1 and Bcl-2 protein expressions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Nuclear morphology of osteoblast cells\u003c/h2\u003e \u003cp\u003eNuclear imaging can be used as a non-destructive molecular visualization tool to know the effect or cytotoxicity caused by the phytomolecules at cellular level. Nuclear morphological abnormalities are considered an essential diagnostic feature to distinguish normal cells from malignant cells. Alterations in nuclear shape of cancer cells can affect transcriptional activity of the cell and thereby gene expressions. In the present study, significant morphological alterations have been observed in the cell nuclei of \u003cem\u003eA. muricata\u003c/em\u003e EAL concentrate treated osteoblast cells compared to untreated control cells. Hoechst dye stains the nuclei of the cells regardless of their viability (Baxter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) and enables them to distinguish the changes that has happened in the morphology of the nuclei induced by the EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e. Morphology of normal osteoblast cells are spindle-elongated with long lamellipodia (Bielack et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Also, the untreated/control osteoblast cells retained its original shape and parallel orientation. Interestingly, the EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e treated cells exhibited nuclei condensation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. An increase in number of cleaved nuclei has also been observed in the cells treated with EAL concentrate above 10 \u0026micro;g/mL concentration. Cells treated with 25 \u0026micro;g/mL concentration of EAL concentrate exhibited typical characteristics of apoptosis such as cell shrinkage and fragmentation, segregated bodies and cell decrement. A decrease in cell viability along with nuclear imaging results confirmed the inhibitory or anti-proliferative effect of EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e on MG-63 osteosarcoma cells\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn the present study, 28 leaf phytochemicals identified from \u003cem\u003eA. muricata\u003c/em\u003e with known concentration of annonacin content obtained through ethyl acetate has been assessed for its efficacy against osteosarcoma. The \u003cem\u003ein-silico\u003c/em\u003e molecular docking analysis of 28 leaf phytochemicals from \u003cem\u003eA. muricata\u003c/em\u003e resulted in identification of three hit bioactives namely, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone, linoleic acid and annonacin to be effective against the protein target, PDGFRA of osteosarcoma with a good binding affinity. Amongst the three hit compounds, 2\u0026rsquo;- hydroxy-5\u0026rsquo;-methyl chalcone satisfied the Lipinski\u0026rsquo;s rule of five and ADMET prediction, while linoleic acid and annonacin showed some violations against the rule of five due to their poor aqueous solubility. Though annonacin showed low drug likeness among the three hit compounds due to its lipophilicity, interestingly it showed good binding energy with PDGFRA by forming two hydrogen bonds at the active site of the target protein. On comparing the molecular dynamic behavior of the three hit compounds with target protein (5k5x), annonacin is found to be more stable. Likewise, \u003cem\u003ein-vitro\u003c/em\u003e cytotoxicity and nuclear imaging with EAL concentrate of \u003cem\u003eA. muricata\u003c/em\u003e treated cells proved its inhibitory action on MG-63 osteosarcoma cells at all studied concentrations. Our \u003cem\u003ein vitro\u003c/em\u003e results supports the chemistry of \u003cem\u003ein-silico\u003c/em\u003e prediction about the effectiveness of leaf phytochemicals of \u003cem\u003eA. muricata\u003c/em\u003e against osteosarcoma. Furthermore, our findings will help researchers to hasten the process of purifying and developing the most efficient therapeutic agent against osteosarcoma from the identified hits or the EAL concentrate without side effects.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that data supporting the findings of this study are available within the article inclusive of supplementary information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have their consent to publish the present work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHS conceptualized and planed the study. HS sourced, extracted and characterized leaf extract of soursop used in this study at CANT, TNAU. CR, KA and JM carried out molecular interaction studies at CPMBB, TNAU. RN and RS carried out \u003cem\u003ein vitro\u003c/em\u003e anti-proliferative activity in MG-63 cancer cells at PSG IAS, Coimbatore. HS drafted the final version of the manuscript and all the authors approved the same.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlali FQ, Liu XX, McLaughlin JL (1999) Annonaceous acetogenins: recent progress. J Nat prod 62: 504-40.\u003c/li\u003e\n\u003cli\u003eBaxter L, Frauchiger V, Textor M, Ap Gwynn I, Richards R (2002) Fibroblast and osteoblast adhesion and morphology on calcium phosphate surfaces. 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J periodon 73:1494-506.\u003c/li\u003e\n\u003cli\u003eBonneau N, Cynober T, Jullian JC, Champy P (2017) 1H qNMR quantification of Annonaceous acetogenins in crude extracts of \u003cem\u003eAnnona muricata\u003c/em\u003e L. Fruit Pulp. Phytochem Anal 28:251-56.\u003c/li\u003e\n\u003cli\u003eDallakyan S, Olson AJ (2015) Small-molecule library screening by docking with PyRx, Chemical biology. Springer pp: 243-50.\u003c/li\u003e\n\u003cli\u003eDe Sousa OV, Vieira GD, de Jesus RG de Pinho J, Yamamoto CH, Alves MS (2010) Anti nociceptive and anti-inflammatory activities of the ethanol extract of \u003cem\u003eAnnona muricata\u003c/em\u003e L. leaves in animal models. Int J Mol Sci 11: 2067-2078.\u003c/li\u003e\n\u003cli\u003eFerguson WS, Goorin AM (2001) Current treatment of osteosarcoma. Clin Cancer investig J 19: 292-315.\u003c/li\u003e\n\u003cli\u003eIsakoff MS, Bielack SS, Meltzer P, Gorlick R (2015) Osteosarcoma: current treatment and a collaborative pathway to success. 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Nucleic Acids Res 46: W363-W67.\u003c/li\u003e\n\u003cli\u003eWilson EA, Russu WA, Shallal HM (2018) Preliminary \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e investigation of a potent platelet derived growth factor receptor (PDGFR) family kinase inhibitor. Bioorg Med Chem Lett 28:1781-84.\u003c/li\u003e\n\u003cli\u003eYang C, Gundala SR, Mukkavilli R, Vangala S, Reid MD, Aneja R (2015) Synergistic interactions among flavonoids and acetogenins in Graviola (\u003cem\u003eAnnona muricata\u003c/em\u003e) leaves confer protection against prostate cancer. Carcinogenesis 36 pp: 656-665.\u003c/li\u003e\n\u003cli\u003eZhou X, Liu H, Zhang M, Li C, Li G (2021) Spectrum‐effect relationship between UPLC fingerprints and anti‐lung cancer effect of Panax ginseng. Phytochem Anal 32:339-46.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Soursop leaves, Annona muricata, GC-MS chemoprofiling, Annonacin, Osteosarcoma, PDGFRA, Molecular docking, ADMET analysis, MG-63 cell lines, Nuclear imaging","lastPublishedDoi":"10.21203/rs.3.rs-2951478/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2951478/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSoursop (\u003cem\u003eAnnona muricata\u003c/em\u003e) is being used in treating various types of cancers and there is no report on effect of soursop leaf phytochemicals against osteosarcoma. Current study identified 28 metabolites from ethyl acetate leaf (EAL) extract through GC-MS chemoprofiling and subjected to \u003cem\u003ein silico\u003c/em\u003e\u0026nbsp;analysis against the potential protein target, Platelet Derived Growth Factor Receptor α (PDGFRA) of osteosarcoma, including Absorption, Distribution, Metabolism, and Excretion and Toxicity\u0026nbsp;(ADMET) analysis to identify possible hit compounds. This resulted in three hit leaf bioactives namely, 2’- hydroxy-5’-methyl chalcone, linoleic acid and annonacin showing good binding affinity with a docking score of -7.4, -7.0 and – 6.9 kcal/mol respectively. With ADMET analysis, 2’- hydroxy-5’-methyl chalcone and linoleic acid obeyed Lipkinsi’s rule of five, whereas annonacin showed slight violation. Among the three docked complexes, annonacin exhibited good stability during molecular dynamic simulation performed with PDGFRA. Hence, concentration of the key marker compound, annonacin in EAL concentrate is found to be 5.032± 0.13 mg/g of leaf sample. Further, EAL concentrate exhibited cytotoxicity (IC\u003csub\u003e50\u003c/sub\u003e value) on MG-63 osteosarcoma cells \u003cem\u003ein vitro \u003c/em\u003efor concentrations ranging from\u0026nbsp;10 to 25 µg/mL and nuclear imaging of osteoblast cells treated with EAL concentrate at 25 µg/mL\u0026nbsp;concentration exhibited typical symptoms of apoptosis. \u003cem\u003eIn vitro \u003c/em\u003ecytotoxicity along with nuclear imaging confirmed EAL concentrate from soursop to be a potential drug candidate in developing new\u0026nbsp;anti-cancer agent against osteosarcoma.\u003c/p\u003e","manuscriptTitle":"Anti-proliferative effect of leaf phytochemicals of soursop (Annona muricata L.) against human osteosarcoma in vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-31 14:24:47","doi":"10.21203/rs.3.rs-2951478/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-05-30T07:37:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-05-29T18:05:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Chemical Papers","date":"2023-05-20T07:36:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-19T13:36:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chemical Papers","date":"2023-05-18T05:40:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chemical-papers","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"chpa","sideBox":"Learn more about [Chemical Papers](http://link.springer.com/journal/11696)","snPcode":"11696","submissionUrl":"https://www.editorialmanager.com/CHPA/default.aspx","title":"Chemical Papers","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99b1f8dd-772d-4b9c-9b21-fd61382b41cd","owner":[],"postedDate":"May 31st, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-26T15:14:04+00:00","versionOfRecord":{"articleIdentity":"rs-2951478","link":"https://doi.org/10.1007/s11696-024-03349-x","journal":{"identity":"chemical-papers","isVorOnly":false,"title":"Chemical Papers"},"publishedOn":"2024-02-23 15:02:08","publishedOnDateReadable":"February 23rd, 2024"},"versionCreatedAt":"2023-05-31 14:24:47","video":"","vorDoi":"10.1007/s11696-024-03349-x","vorDoiUrl":"https://doi.org/10.1007/s11696-024-03349-x","workflowStages":[]},"version":"v1","identity":"rs-2951478","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2951478","identity":"rs-2951478","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}