Isolation and biological evaluation 7-hydroxy flavone from Avicennia officinalis L: insights from extensive in vitro, DFT, molecular docking and molecular dynamics simulation studies

Abstract The flavonoid based 7-hydroxy flavone (PubChem CID: 5281894; molecular formula: C15H10O3) molecule has been isolated for the first time from the methanolic extract from the leaves of Avicennia officinalis L. in the tropical mangrove ecosystem of Andaman and Nicobar Islands (ANI), India. The molecular structure of bioactive compound was characterized by spectroscopic analysis, including FT-IR, 1H, 13C NMR spectroscopy and ESI-HRMS and elucidated as 7-hydroxy flavone. An anticancer activity of isolated 7-hydroxy flavone was evaluated by in vitro study against two different human cancer cell lines namely, HeLa (cervical cells) and MDA-MB231 (breast cells) and they exhibited promising anticancer activity with IC50 values are 22.5602 ± 0.21 µg/mL and 3.86474 ± 0.35 µg/mL, respectively. The antioxidant property of 7-hydroxy flavone at a standard concentration of 50 µg, was found to be (IC50) 5.5486 ± 0.81 µg/mL. In summary, this investigation provides evidence that 7-hydroxy flavone exhibits both anticancer and antioxidant properties. Meanwhile, the antimicrobial activity ability of 7-hydroxy flavone were also evaluated using three Gram positive and two Gram negative strain exhibited no antimicrobial activities. Density-functional theory (DFT) studies confirm the structure is global minima in the PES, from the optimized geometry FMO and MESP map analyzed. Further, the molecular docking and molecular dynamics simulation studies result shows that 7-hydroxy flavone has the better binding ability with anti-apoptotic Bcl-2 protein with the estimated free energy of binding of –6.3 kcal/mol. This bioactive compound may be act as drug candidate for treating various kinds of cancers. Highlights A 7-hydroxy flavone molecule has been isolated from Avicennia officinalis. The isolated pure compound was subjected to spectral analysis such as FT-IR, 1H NMR, 13C NMR spectral data and HRMS analysis for skeleton of the molecule. The anticancer activity of 7-hydroxy flavone studied against Cervical (HeLa) cancer cell lines and breast (MDA-MB231) cancer cell lines with the IC50 values of 22.5602 ± 0.21 µg/mL and 3.86474 ± 0.35 µg/mL), respectively. The antioxidant properties of 7-hydroxy flavone were found to be (IC50) 5.5486 ± 0.81 µg/mL at a standard concentration of 50 µg. DFT, molecular docking and MD simulation results explained that 7-hydroxy flavone could be the most promising candidate to inhibit the function of anti-apoptotic Bcl-2 protein in cancerous cell. Communicated by Ramaswamy H. Sarma

However, the mangrove plants in the Andaman and Nicobar Islands (ANI), one of the most ecologically diverse places in the tropical region, have not been chemically and biologically studied in detail (Sachithanandam et al., 2019(Sachithanandam et al., , 2020. Minimal research has been carried out towards the isolation of flavonoids compounds such as hydroxyl methyl flavone (1), velutin (2), quercetin (3), kaemferol (4), luteolin (5) (Figure 1) from A. officinalis mangrove plants (Anjaneyulu et al., 2003;Ghosh et al., 2019;Kar et al., 2014;Kaliamurthi & Selvaraj, 2016;Majumdar et al., 1981;Sachithanandam et al., 2020). In this regard, we studied leaves of A. officinalis from ANI mangroves to explore the possibilities of isolating novel compounds with promising antioxidant and anticancer activities. We had isolated active fraction, namely, 7-hydroxy flavone (6) Molecular Formula: C 15 H 10 O 3 (Figure 1), when the A. officinalis leaves were subjected to methanol extraction followed by convention column chromatography. Flavonoids are an essential class of polyphenolic compounds containing two benzene rings (A and B) connected via a heterocyclic pyrone ring (C). The 7hydroxyl flavonoid in which the flavone nucleus is substituted at position seven by a hydroxy group (Sharma & Janmeda, 2017). In addition to 7-hydroxy flavone, its derivatives have exhibited diverse biological activities as an antioxidant, antimicrobial, anticancer, anti-fungal and antidiabetic activities (Assaw et al., 2020;Kishore et al., 2018;Nisar et al., 2019;Thatoi et al., 2016). Herein, we present the isolation, structure characterization, biological evaluation of these metabolites.

Plant material
Raw and fresh leaves of a mangrove plant, A. officinalis (Figure 2), were collected from the Sippighat mangrove area, South Andaman Island, India, in September 2018. The leaves were washed thoroughly with deionized water, dried at room temperature, and then pulverized into a fine powder using a mixer grinder.

Chemicals, reagents and standards
All Chemicals, organic solvents, and reagents were purchased from Finar, Merck, Himedia Pvt. Ltd., Sigma, India. Glass plates coated with silica gel of 60-120 mesh employed for thin-layer chromatography (TLC) was obtained from Merck chemicals. Organic solvents of analytical grade: Ethyl acetate, hexane, acetone, and methanol were also obtained from Merck chemicals. The stock solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was prepared at a concentration of 0.1 mM, and other standard solutions were prepared at the concentration of mg/mL. Apart from this, all glassware used in the experiments were washed, rinsed with double distilled water, and dried in an oven at 250 C.

Instruments
The FT-IR, NMR and HRMS analysis were carried out for the structural characterization and elucidation of the bioactive compound. FT-IR analysis was performed using PerkinElmer Frontier spectrometer which identifies the significant chemical bond and functional group present in the phytochemical extract. In NMR analysis, 1 H-NMR (400 MHz and 500 MHz) and 13 C-NMR (100 and 125 MHz) were recorded on Bruker NMR Spectrometer (Avance II, Bruker Corporation, USA). The NMR spectra were recorded by using DMSO-d 6 deutrated solvent at room temperature. The 1 H-NMR chemical shifts (DH) and 13 C-NMR chemical shifts (DC) were mentioned as parts per millions Parts per millions (PPM) whereas, downfield from trimethylsilane and coupling constants (J) were mentioned in terms of Hertz (Hz) Parthiban & Makam, 2020;Sachithanandam et al., 2020). Abbreviated forms of NMR data, i.e., chemical shift, multiplicity, coupling constant and integration are represented as s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet) and sxt (sextet). The HRMS analysis provides in house library of numerous compounds that could allow identification of the desired bioactive compound. The UV-visible spectra in the experiments were obtained using Agilent Cary 100 spectrometer. An accurate melting point and melting range of the bioactive compound was determined using VEEGO VMP-D/DS precision digital melting/boiling point apparatus.

Extraction and isolation
The extraction of bioactive component details described our recent study (Sachithanandam et al., 2020, obtained as a fine powder (10 g) mixed with high polarity organic solvent  like methanol (300 mL), was done in a conical flask and kept overnight with increasing polarity at ambient temperature (27 C). It was incubated at room temperature for 48 hours at 150 rpm in an orbital shaker (Thermo Fisher Scientific). The extracts were filtered with Whatman No. 1 filter paper. Each supernatant of the extract was concentrated/dried in vacuo using a rotary evaporator (CYBER, Germany) under reduced pressure at 40 C temperature to afford 2 g of crude components. 2 0 ,4 0 ,5-Trihydroxy-5 0 ,6,7trimethoxyflavone (arcapillin)

Purification of bioactive compound 7-hydroxy flavone
Based on the in vitro assay, the crude extract of A. officinalis (1 g) was dissolved in about 0.5 mL of dichloromethane (DCM) and analytical TLC (20: 80, methanol: chloroform) was performed on precoated aluminium sheets of silica gel G/UV-254 of 0.2 mm thickness (Merck, Germany). Plates were observed under short wavelength UV light and showed one active spot with few low intensive multiple spots. The retention factors (R f ) of intensive spot R f values are 0.7. The methanolic crude extract (1 g) were subjected into column chromatography, which were packed with 100% chloroform solvent on a silica gel (100-200 mesh). Then extracts were eluted with 10-100% mixture of Chloroform: Methanol to provide multiple fractions according to TLC. The active fraction upper spot (R f ¼ 0.7) were collected at 60% chloroform solvent mixture (60% chloroform: 40% Methanol). These fractions collected through test tube and concentrated/dried in vacuo by using rotary evaporator (CYBER, Germany) under reduced pressure to get coloured product. The isolated compound further purified by slow evaporation method by using mixture of dichloromethane and hexane (90% DCM: 10% hexane) to afford pure orange red coloured 7-hydroxy flavone with yield of 5% (50 mg: mol. wt. 240.00; m.p. 200-202 C). The remaining low intensive fractions have not been characterized due to low availability and less concentrations. Further, the structure of the isolated pure compound was characterized by spectral analysis such as FT-IR, 1 H NMR, 13 C NMR spectral data and HRMS analysis. Human cervical (HeLa) cancer and breast (MDA-MB231) cancer cell lines were obtained from National Centre for Cell Sciences, Pune and maintained in Dulbecco's modified eagle medium (DMEM). The medium was supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillinstreptomycin and 0.1% amphotericin. Cell lines were maintained at 37 C in a humidified CO 2 (5%) incubator (Formasteric cycle, Thermo Scientific).

Antimicrobial assay
Inhibition of bacterial growth in response to the antibacterial activity exhibited by the crude extracts of mangrove plant samples were assessed by agar well diffusion assay (Zheng et al., 2005). As per McFarland standard solution, exponential phase pathogenic bacterial inoculums (10 8 cells/mL) were prepared and swabbed onto sterile Muller-Hinton agar plates. Then the wells (6 mm in diameter) were made using cork-borer. Dried crude methanol extracts were dissolved in dimethyl-sulfoxide (DMSO) at 5 mg/mL concentration. To each well, 100 mL of methanol extract from ten different mangrove plants were added individually and incubated at 37 C for 24 h. In this assay, 100 mL of antibiotic-gentamycin at mg/mL concentration was added as positive control for both Gram-positive and negative bacteria strains; while 100 mL of pure DMSO was added to well as negative control (Lalitha et al., 2016;Sachithanandam et al., 2020). Percentage of antibacterial activity exhibited by crude extracts were measured based on zone of inhibition. The extracts that efficiently inhibited the bacterial growth was further assessed for minimum inhibitory concentration (MIC). Each sample was done in triplicates.

Antioxidant assay
This in vitro free radical scavenging assay involves 0.1 mM DPPH solution dissolved in 95% methanol and different concentrations of varied mangrove leaf methanol extracts (1, 10, 50, 100, 150, 200 mg/mL); where antioxidant activity of crude extracts will be determined spectrophotometrically. To 1 mL of DPPH solution, 1 mL of crude extracts at different concentrations were added and allowed to stand for 30 min at room temperature in dark. Then the absorbance of the reaction mixture was measured at 517 nm against methanol as blank (Sachithanandam et al., 2020). Comparing ascorbic acid as standard, percentage of scavenging inhibition was determined using the formula, where, A c is the absorbance of control and A s is the absorbance of sample. Each sample was done in triplicates.

Cytotoxicity assay
Cytotoxic activity of the bioactive compound (characterized above) was assessed against Cervical (HeLa) and Breast (MDA-MB231) cancer cell lines by performing MTT cell viability assay (Sachithanandam et al., 2020). This assay involves the treatment of HeLa and MDA-MB231 cancer cell lines with bioactive compound at varied concentration (0.5, 5, 25, 50 and 100 mg/mL) dissolved in DMSO. Then in presence of metabolically active cells, MTT (3,4,5-(dimethyl-thiazol-2-yl) 2-5-diphenyl tetrazolium bromide) is reduced to purple coloured formazan crystals (Lalitha et al., 2016). Initially, the cell lines were (0.2 Â 10 5 cells) were seeded in 96-well plates and incubated overnight with 200 mL of the medium. Following this, the resulting monolayer of cells (70% confluence) were treated with different concentrations of purified bioactive compound and incubated for 24 h at 37 C. In this experiment, cyclohexamide (5 mM) was used as positive control and the growth medium without cell culture was used as negative control. The culture medium was replaced with fresh medium (200 mL), 10 mL of MTT was added to each well, incubated again for 4 h at 37 C and the medium was aspirated carefully. The remaining formazan crystals were then solubilized with 50 mL of 0.05 M of HCl and the absorbance was measured at 570 nm. The percentage of cell viability was calculated using the formula, where, A T0 is the absorbance of the untreated cells (negative control; considered as 100% viable) and A T is the absorbance of treated cells. IC 50 was determined based on regression analysis of dose response curve.

DFT studies
7-Hydroxy flavone was optimized with B3LYP functional (Becke, 1993) 6-31 G Ã level (Ditchfield et al., 1971;Hariharan & Pople, 1973;Hehre et al., 1972) of DFT. From the optimized geometry further computed for vibrational analysis in order to confirm the global minima in the potential energy surface (PES). Further, we have also analyzed Frontier molecular orbital (FMO) analysis and Molecular electrostatic potential mapping (MESP) analysis on optimized geometry. All these computation was carried out by using the Gaussian 09 program package (Frisch et al., 2009).

Molecular docking and molecular dynamics simulation studies
The molecular docking study of flavone compound with target anti-apoptotic Bcl-2 protein was carried out by Auto Dock Vina (Trott & Olson, 2010). The three-dimensional structure of Bcl-2 protein (PDB ID: 1GJH) was retrieved from Research Collaboratory Structural Bioinformatics -Protein Data Bank (RCSB-PDB), which is chosen for the drug target against 7-hydroxy flavone compound due to the abnormal expression of anti-apoptotic Bcl-2 protein in most of the cancers (Dhamodharan et al., 2018;Sachithanandam et al., 2020). The Bcl-2 family of proteins are considered as a crucial drug target for most of the cancers (Ramos et al., 2019). Once we identified the drug target protein, the protein and ligand preparation steps were carried out to make both the molecules for further molecular docking and molecular dynamics studies. The protein preparation steps include (a) addition of polar hydrogens, (b) addition of Kollmann partial charges, (c) merging of non-polar hydrogens and (d) finally saved into PDBQT (coordinates þ partial charges þ atom type) format. Similarly, the ligand preparation step includes (a) addition of polar hydrogens, (b) merging of non-polar hydrogens, (c) addition of gasteiger charges and (d) finally saved into PDBQT format. After that, the receptor grid map was generated around the binding groove or binding cleft of anti-apoptotic Bcl-2 protein with the following parameters: center: . The site-specific or direct docking calculation was performed between the binding cleft of Bcl-2 protein and the 7-hydroxy flavone molecule using Auto Dock Vina (Trott & Olson, 2010). The best protein-ligand complex was chosen based on various structural parameters namely estimated binding free energy (DG), estimated inhibition constant (Ki), and the higher number of docking orientations or docking solutions present at the binding cleft of Bcl-2 protein. Finally, the analysis and visualization of the docking results were performed by using three programs namely PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schr€ odinger, LLC.), MGLTools (http://mgltools. scripps.edu/) (Morris et al., 2009) and LigPlot (Wallace et al., 1995) programs respectively. Apart from molecular docking calculation, we also performed molecular dynamics (MD) simulation of unbound, and 7-hydroxy flavone bound Bcl-2 structures with the time period of 50 ns using Gromacs Version 2019 (Van Der Spoel et al., 2005). The MD simulation begins with Gromacs formatted input or GRO, topology (TOP) and positional restraint (POSRE) files from the input PDB file. After that, the cubic box was generated around unbound 7-hydroxy flavone bound Bcl-2 structure. Then, the Single point charge extended (SPCE) water model was used to add the water molecules (Table 2) into the box. The counterions (positive or negative charges) were added (Table 2) to the system in case if they did not attain the neutral charge otherwise this step can be excluded. Once the system achieved the neutral or non-zero charge, energy minimization using steepest descent approach followed by equilibrations were performed at two different steps (NVT and NPT) with the time period of 100 ps and then finally production MD simulation was performed with the time period of 50 ns.
For the MD simulation of ligand bound Bcl-2 structure, initially, the topology (ITP) and GRO files of 7-hydroxy flavone molecule was generated using web-based topology builder namely PRODRG (Sch€ uttelkopf & van Aalten, 2004), after that, MD simulation steps were carried out on 7-hydroxy flavone bounded Bcl-2 structure with the modifications in the molecular dynamics parameters (MDP) files of energy minimization, NVT, NPT equilibrations and production MD steps. Finally,

Characterization of 7-hydroxy flavone
The obtained purified flavonoid compound was characterized on the basis of FT-IR, 1 H NMR, 13 C NMR spectral data and HRMS analysis as described in a previous work (Aksnes et al., 1996;Kostrzewa-Susłow & Janeczko 2012). The IR absorption spectrum of isolated compound showed absorption peaks at 3292 cm À1 indicated OH group present in the molecule. Peak absorptions at 1606 cm À1 indicated carbonyl group and 1510 cm À1 indicated C ¼ C peak in the molecule (supplementary material Figure 1). The 1 H NMR (400 MH Z ) spectrum of 7hydroxy flavone displayed a characteristic broad signal for OH for phenolic at 8.05 ppm at a singlet. The signals located at D 7.92 as triplet, 7.65 7.65 as doublet of doublet and 7.00 ppm as doublet of doublet attributable to aromatic CH proton in the A ring. The C-3 active hydrogen located at 6.48 as multiplet in the C ring. The signals located at D 7.46 as doublet and 7.65 as doublet of doublet ppm indicated to aromatic CH proton in the B ring (supplementary material Figure 2). The 13 C NMR (100 MHz) spectrum of 7-hydroxy flavones displayed the estimated fifteen carbon signals. The carbonyl carbon appeared at 202.74 (CO) ppm in the structure. The quaternary carbons appeared at 164.81 at C-2, 168.09 at C-7, 164.81 at C-9, 115.87 at C-10, 136.28 at C-11 ppm. The aromatic CH carbons appeared at 126.68 at C-5, 112.79 at C-6, 107.37 at C-8, 129.58 at C-12, 128.44 at C-12 0 , 134.57 at C-13,133.88 at C-13 0 , 131.45 at C-14 ppm (supplementary material Figure 3). The detailed NMR Results analyses are given in Table 3. The mass of the purified 7-hydroxy flavone was determined as 238.2420. ESI-HRMS of C 15 H 10 O 3 was 239.0701(supplementary material Figure 4). The combined FT-IR, NMR and HRMS spectral study confirmed that the compound was identified as 7-hydroxy flavone similar to those reported by (Aksnes et al., 1996;Edyta & Janeczko, 2002).

Antioxidant activity of 7-hydroxy flavone
The free radical scavenging activity of the isolated bioactive compound has also been elucidated based on scavenging activity of the stable DPPH assay (Figure 3). The antioxidant activity of 7-Hydroxy flavone isolated from methanolic leaf extract of A. officinalis, was carried out with different doses, namely 1, 10, 25, 50, 100, 150 and 200 mg/mL, respectively. The results confirmed, a dose dependent inhibitory activity of 7-hydroxy flavone revealed free radical scavenging activity was found. Briefly, the DPPH turns yellow from purple an indicate presence antioxidant activity. The 7-Hydroxy flavone was showed less than 50%, respectively. However, ascorbic acid was used as positive control exhibited better radical scavenging effect IC 50 value of 5.5486 ± 0.81 mg/mL respectively (Figure 3). In summary, this study provides evidence that 7-Hydroxy flavone exhibit interesting antioxidant activity expressed. Similarly earlier work also supported to the present investigation (Jasril et al., 2003;Kikuzaki & Nakatani, 1993;Lee et al., 1998). Figure 3 depicted that kinetic activities of different concentration and estimated the antioxidant activity of maximum reaction time. The higher scavenging activity of the particular concentration of 7-Hydroxy flavone may be assumed that -OH groups are play a major role in electron transfer reaction to DPPH, which attributed to the highly significant contribution of 7-hydroxy flavone compound (Alaklabi et al., 2018).

Cytotoxicity of 7-hydroxy flavone
Anti-cancer activity of 7-hydroxy flavone isolated from mangrove plant (A. officinalis) was determined by MTT reduction assay on cervical (HeLa) and breast (MDA-MB231) cancer cell lines. The results confirmed that the dose-dependent inhibitory activity of 7-hydroxy flavone revealed cytotoxicity activity against HeLa and MDA-MB231cancer cell lines with the IC 50 concentration of HeLa (22.5602 ± 0.21 mg/mL) and MDA-MB231 (3.86474 ± 0.35 mg/mL), respectively (Figure 4). Based on the previous study values of IC 50 < 10 mg/mL are categorized as "very active"; IC 50 between 10 À 20 mg/mL are categorized as "active"; and IC 50 between 20-100 mg/mL are categorized as "quite active" in mangrove plants (Murakami et al., 1998;Jasril et al., 2003;Sachithanandam et al., 2020Sachithanandam et al., , 2021.The 7-hydroxy flavone is more cytotoxic against MDA-MB231 cells compared HeLa cells. All the studied Cancer cell line exhibited strong inhibitory activity ability of 7hydroxy flavone. Similarly, anticancer against HeLa cells (Sachithanandam et al., 2020 and Colon (Widr) cancer   cells showed quite active with IC 50 value of 25.73 lg/mL and 83.75 lg/mL, respectively and was quite active than the present study HeLa (22.5602 ± 0.21 mg/mL) data. Similarly, Jasril et al. (2003) and Middleton et al. (2000), reported that 7hydroxy flavone responsible for several biological activities like antitumor, anti-inflammatory, antiallergic action. The 7hydroxy flavone more active cytotoxic effects on breast cancer cells MDA-MB231 (IC 50 ¼ 3.86474 ± 0.35 mg/mL) than on HeLa cells IC 50 ¼ 22.5602 ± 0.21 mg/mL. The results of present investigation may also support the justification on the traditional use of this mangrove plant as a remedy for swelling.

Density-functional theory (DFT) studies
The optimized structure of 7-hydroxy flavones is shown in Figure 5. And important bond lengths are highlighted in the structure, such as C-N, C-O, C-C and O-H. Vibration   calculation was done to ensure the structure is the global minima on the potential energy surface. And the functional group was confirmed by their corresponding vibrational frequencies which is very closer to the experimental values. The frontier molecular orbitals, the energy gap between the HOMO and LUMO orbitals of the 7-hydroxy flavone is 4.60 eV. The positive lopes are shown in red colour and the negative phase is represented in green colour, are shown in Figure 6. The mapping of ESP against the total electron density surface simultaneously displays electrostatic potential (electron p nuclei) distribution, size, dipole moments and molecular shape, of the molecule and it helpful to identify with the relative polarity pictorially. The MESP surfaces of the molecules are created by same level of theory and method. The total electron density mapped with the electrostatic potential surface of 7-hydroxy flavone is shown in Figure 7. The colour of MESP surface is red, which indicates that region is an electron-rich, partially negative charge; yellow means slightly electron-rich region; green indicates the neutral; blue colour indicates that electron-deficient, partially positive charge; light blue means slightly electron-deficient region; respectively. The region around the hydroxyl group represents the most positive potential region (blue) and the more negative charge around the carbonyl group. The majority of green region is found in the MESP surfaces on the 7-hydroxyflavone.

Molecular docking studies
Molecular docking calculation was performed between 7hydroxy flavone and anti-apoptotic Bcl-2 protein with the estimated free energy of binding (DG) of -6.3 kcal/mol. This molecule is oriented in binding groove or binding cleft of target protein. The binding groove of Bcl-2 protein was formed by three essential functional domains BH1, BH2 and BH3 and the groove was stabilized by fourth domain BH4. The electrostatic surface potential map (Figure 8) revealed that most of the region of 7-hydroxy flavone molecule is oriented in the negatively charged amino acid region of Bcl-2. The ligand molecule does not form any hydrogen bonds with the target protein whereas it forms several hydrophobic interactions with key residues of binding groove namely Thr55, Gln58, Ala59, Asp62, Phe63, Val107, Tyr161 and Pro163 (Figure 8). The interacting residues are mostly oriented in the binding cleft of anti-apoptotic Bcl-2 protein.
In the present study, we have employed Venetoclax (Li et al., 2019) as a control molecule because it is a known and more specific inhibitor for anti-apoptotic Bcl-2 protein. We have (Ramos et al. (2019) previously reported the molecular docking and molecular dynamics simulation of Venetoclax (or control molecule) towards both chimeric and physiological form of anti-apoptotic Bcl-2 protein. In comparison with 7hydroxy flavone (DG: -6.3 kcal/mol) and our previously reported molecules namely Quinizarin (DG: -6.2 kcal/mol; Sachithanandam et al., 2021), Quercetin (DG: -6.9 kcal/mol; Sachithanandam et al., 2020) and Gallic acid (DG: -5.4 kcal/ mol; Sachithanandam et al., 2020), the control molecule showed strong and higher binding affinity (-10.24 kcal/mol for the physiological Bcl-2 and -11.35 kcal/mol for the chimeric form) towards anti-apoptotic Bcl-2 protein. Because this control molecule occupied the entire region of binding cleft of target protein since the binding cleft of anti-apoptotic is extended. Although a control molecule showed higher binding affinity, the presented lead candidate, 7hydroxy flavone is also showing appreciable binding affinity (-6.3 kcal/mol) towards target Bcl-2 protein. Thus, the proposed in silico interaction study hypothesized that 7-hydroxy flavone could be the BH3 mimetics as gallic acid, quinizarin, quercetin and venetoclax.
To evaluate the results obtained from molecular docking calculation of 7-hydroxy flavone with anti-apoptotic Bcl-2 protein, we subsequently performed comparative molecular dynamics simulation of unbound and 7-hydroxy flavone bound anti-apoptotic Bcl-2 structures. The MD simulation results (Table 4 and Figure 9) explained that upon interaction of ligand molecule to the binding cleft of anti-apoptotic Bcl-2 protein which does not significantly alter the structural stability, structural integrity, and structural compactness of Bcl-2 protein with the results obtained from global as well as essential motion (based on Principal Component Analysis) analysis.
Moreover, the trace of covariance matrix values of both free and ligand bounded Bcl-2 structures almost closer to each other which indicate that after the binding of ligand to the binding cleft of Bcl-2 protein does not significantly alter or deviate the structural stability. The average RMSD and RMSF values of both free and 7-hydroxy flavone bound Bcl-2 structures are almost same. Similarly, the RMSD and RMSF pattern of free and ligand bound Bcl-2 structures are nearly consistent with each other. Interestingly, the average of Rg and SASA values of ligand bound structure has lesser values which indicate that 7-hydroxy flavone molecule enhances the compactness folding properties of target protein moreover it also maintains the compactness of the protein.
From the hydrogen bond analysis, it has been observed that maximum number of four intermolecular hydrogen bonds formed consistently between protein and ligand molecule which indicate that the ligand molecule binds strongly to the binding cleft of Bcl-2 protein. Together the results obtained from both molecular docking and molecular dynamics simulation (Table 4 and Figure 9), this molecule could be a most promising lead candidate for further anticancer drug designing and development process. In order to understand the binding affinity between Bcl-2 and 7-hydroxy flavone during MD simulation, we have performed MM-PBSA binding free energy analysis based on stable trajectories (40 to 50 ns) of protein-ligand complex using g_mmpbsa package (Kumari et al., 2014). From the results of MM/PBSA binding free energy analysis, it has been observed that the natural ligand showed stronger binding affinity towards the binding groove of anti-apoptotic Bcl-2 protein with the binding free energy of -141.405 ± 10.948 kJ/mol (-33.8 kcal/mol) ( Figure  10). The negative binding free energy value concludes that the 7-hydroxy flavone molecule binds strongly to the target protein at the same time it does not affect the structural stability of anti-apoptotic Bcl-2 protein.
To examine the ligand induced conformation changes, we have subsequently performed domain distance and secondary structure analysis between unbound and 7-hydroxy bound Bcl-2 structures. From the results of domain distance analysis, interestingly, it has been observed that the distance values between each domain of Bcl-2 protein is changed because the ligand molecule enhances the conformational changes of target protein. Particularly, the distance value between BH4-BH3, BH4-BH2, BH4-BH1, BH3-BH2 (Table 5) got reduced indicate that ligand occupied strongly towards the binding groove of anti-apoptotic Bcl-2 protein since the binding groove is formed by BH1, BH2 and BH3 domains and it is stabilized by BH4 domain. Similarly, the secondary structure composition (Figure 11) between unbound and 7hydroxy Bcl-2 structures is also changed indicated that ligand induces the conformational changes of target protein.
Together the results obtained from domain distance and secondary structural analysis, this ligand induced the conformational changes of Bcl-2 protein.

Conclusion
In conclusion, the 7-hydroxyflavone bioactive compound has been successfully isolated from methanolic extract of A. officinalis. We have optimized the structure of 7-hydroxy flavone using the DFT method and predicted their reactive regions from the FMO and MESP analysis. Further, we have used optimized geometry for the molecular docking and molecular dynamics simulation studies, found to have -6.3 kcal/mol of estimated binding free energy with and Bcl-2 protein.
Together the results obtained from both molecular docking and MD simulation, 7-hydroxy flavone molecule could be a most promising lead candidate to inhibit the function of anti-apoptotic Bcl-2 protein in cancerous cells. 7-Hydroxy flavone was found to have remarkable antioxidant with scavenging effect IC 50 value of 5.5486 ± 0.81 mg/mL. The bioactive 7-hydroxy flavone effects anticancer activities with IC 50 3.86474 ± 0.35 and 22.5602 ± 0.21 mg/mL for breast MDA-MB231 cancer cells and cervical (HeLa) cells, with active category as anticancer. The present investigation of anticancer and antioxidant was elucidated using in vitro analysis only and future study for the mechanism of action and in vivo analysis will lead to a more complete assessment of 7hydroxy flavone from A. officinalis leaves extracts.

Acknowledgments
We earnestly acknowledge our gratitude to the Forest Department, for providing sample collection on mangroves of ANI and NCSCM publication committee members who reviewing our article and recommendation for the publication. Views expressed are of the authors only and not necessarily of the affiliated organizations. Authors also acknowledges Department of Chemistry, Pondicherry University and Sharda University for providing computational support. This work has been carried out as a part of the In-house project "Traditional Knowledge of tribal communities in Islands of India with reference to medicinal plants" (Contribution number: NCSCM/PUB/2022/0001).  Figure 11. Secondary structure analysis of unbound and 7-hydroxy bound Bcl-2 structure.