Aconitine linoleate, a natural lipo-diterpenoid alkaloid, stimulates anti-proliferative activity reversing doxorubicin-resistant in MCF-7/ADR breast cancer cells as a selective topoisomerase IIα inhibitor

Aconitine linoleate, a natural lipo-diterpenoid alkaloid, stimulates anti-proliferative activity reversing doxorubicin-resistant in MCF-7/ADR breast cancer cells as a selective topoisomerase IIα inhibitor | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Aconitine linoleate, a natural lipo-diterpenoid alkaloid, stimulates anti-proliferative activity reversing doxorubicin-resistant in MCF-7/ADR breast cancer cells as a selective topoisomerase IIα inhibitor Shangxian Luan, Yingying Gao, Xiaoxia Liang, Li Zhang, Qiang Wu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1005235/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 1 You are reading this latest preprint version Abstract Aconitine linoleate ( 1 ) is a lipo-diterpenoid alkaloid, isolated from Aconitum sinchiangense W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of 1 against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation in vivo . The cytotoxic activities of 1 were measured in vitro. Also, we investigated the latent mechanism of 1 by cell cycle analysis in MCF-7/ADR cells, and Topo I, Topo IIα inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of 1 was detected on mice. 1 exhibited significant anti-tumor activity against both MCF-7 and MCF-7/ADR cells, with IC 50 value of 7.58 and 7.02 µM, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than adriamycin in MCF-7/ADR cell lines. The molecular docking and topo inhibition test found that it's a selective inhibitor of topoisomerase Ⅱα. Moreover, activation of damage response pathway of the DNA leads to cell cycle arrest at G 0 G 1 phase. Furthermore, the in vivo acute toxicity of 1 in mice displayed lower toxicity than aconitine, with LD 50 of 2.2×10 5 nmol/Kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, 1 has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells, and greatly reduces in vivo toxicity, which suggesting this kind of lipo-alkaloids are powerful and promising antitumor compounds for breast cancer. Clinical Pharmacology Aconitine linoleate cytotoxic activity topoisomerase IIα inhibitor acute toxicity MCF-7 and MCF-7/ADR breast cancer cells Molecular modeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction According to the statistical data from the World Health Organization (WHO) in 2020, breast cancer has taken the top one place both in diagnose rate and mortality rate for female (Sung et al., 2021 ). However, its treatment has been hindered by multidrug resistance (MDR), leading to the urge for new antitumor agent targeted specific target, especially for reversing the resistant problems. Among the various anticancer targets, DNA topoisomerases (topos) are well-known ones (Li and Liu, 2001 , Pommier, 2006 , McClendon and Osheroff, 2007 , Nitiss, 2009b , Nitiss, 2009a ). Topos inhibitors are generally classified into Topo I inhibitors as camptothecin (CPT) (Schoeffler and Berger, 2008 ), and Topo II inhibitors as amsacrine, etoposide and doxorubicin (Khadka and Cho, 2013 ). However, the clinic use of current anticancer topoisomerase inhibitors have been limited by some major negative consequences. Firstly, most clinically active topo inhibitors are topo poisons, such as etoposide and doxorubicin, which usually need monitoring for the severe genetic toxicity (Jun et al., 2015 ). Secondly, the severe dose-limiting toxicities and drug resistance of Topo I inhibitors like CPT have been emerged. Thirdly, the secondary malignancies led by the inhibition of Topo IIβ in the clinic use of the unselective Topo II inhibitors have been reported (Felix, 1998 , Mistry et al., 2005 , Azarova et al., 2007 ). For these reasons, many researchers have targeted Topo IIα, rather than IIβ, on developing topo catalytic inhibitors, which induces cancer cell death through the elimination of the essential enzymatic activity, rather than topo poisons(Albert et al., 2013 , Liang et al., 2019 ). As a widely used traditional Chinese medicine, Aconitum possesses anti-inflammatory, analgesic, and anti-cancer activities (Kim et al., 2002 , Liang et al., 2016b ). Single Aconitum and its prescriptions have been documented for the treatment of cancer since last century in China (Mingxin Tang and Sun, 1986, He Shu et al., 2009, Chen, 2015 , Qiuping Xu et al., 2016 , Duan et al., 2018 ). However, the precise mechanisms of the Aconitum eliciting anticancer effects have not yet been fully clarified due to the diverse structures of compounds in Aconitum . Diterpenoid alkaloids, the main active components from Aconitum , have been reported as potential cytotoxic agents for decades (Li et al., 2005 , Hazawa et al., 2009a , Hazawa et al., 2009b , Wada et al., 2011 , Wada, 2012 , Wada et al., 2012 , Wada et al., 2015a , Wada et al., 2015b , Liang et al., 2016a , Liang et al., 2017 , Liang et al., 2018 , Wada K., 2019) (Figure 1 ). But the significant toxicities of diterpenoid alkaloids, such as the neurotoxicity and cardiotoxicity of aconitine ( 4 ), limited their further clinical application. Lipo-diterpenoid alkaloids, a kind of diterpenoid alkaloids bearing long ester groups (such as 1-3 in Figure 1 ), are common components obtained from Aconitum , which have been reported with extensive pharmacological activities and low toxicity (Kitagawa et al., 1982 , Isao et al., 1984 , Hanuman and Katz, 1994 , Yamashita et al., 2018 , Yamashita et al., 2020 ). In 2017, our group first reported the obvious cytotoxic activities of lipo-diterpenoids 1 and 3 , against HL-60, A-549, SMCC-7721, MCF-7 and SW480 cell lines, comparable to cisplatin (Liang et al., 2016a , Liang et al., 2017 ). Later, the good cytotoxic activities of one lipo-alkaloid against several tumor cell lines (A549, MDA-MB-231, MCF-7, KB, KB-VIN) also have been reported by Koji Wada group (Koji Wada, 2019 , Yamashita H., 2020). Then, the highly selective index (SI >10) for compound 1 between MCF-7 and mouse fibroblast cells, has surprised us, arousing a deeper pharmacological mechanism research in current study. In this work, aconitine linoleate ( 1 ) was investigated for its anti-proliferative activity in vitro against MCF-7 and adramycin resistant subline MCF-7/ADR cell lines. In order to explore its mechanistic pathways, the lead compound 1 was performed extra investigations such as cell cycle analysis, topoisomerase I / II inhibition, and molecular docking. Meanwhile, for determining its safety the acute toxicity on mice was also tested. 2. Materials And Methods 2.1 Materials and reagents Aconitine linoleate ( 1 ) (isolated from the Aconitum sinchiangense W. T. Wang, plants were collected from Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China, in 2016, and was identified by Dr. Lixia Li. The plants are Stored in the Natural Medicine Research Center of Sichuan Agricultural University, the voucher specimen No. 20140616-1); MCF-7 cell line (Breast cancer cell MCF-7), MCF-7/ADR (adramycin resistant subline) and NIH3T3 cell line (Mouse fibroblast cells) (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); Kuming mice (CHENGDU DOSSY EXPERIMENTAL ANIMALS), PBS (HyClone); DMEM high glucose (HyClone); fetal bovineserum (FBS) (Gibco); propidium iodide (PI) (Yeasen Biotech); Adriamycin (RYON); Etoposide and hydroxy camptothecin (TargetMol);Trypsin-EDTA (0.25%), phenol red (solarbio); Penicillin-Streptomycin Liquid (solarbio); CellTiter 96®AQueous One Solution Cell Proliferation Assay (Promega); Paraffin wax (Shanghai Hualing kangfu Machinery Factory); Ehrlich hematoxylin stain and Eosin Solution (solarbio); LDH Cytotoxicity Assay Kit (solarbio); Prestained Protein Ladder, 10 to 180 kDa and 10-170 kDa, RIPA Lysis Buffer, Topoisomerase Ⅱ alpha Rabbit Monoclonal Antibody, Topoisomerase Ⅰ Rabbit Monoclonal Antibody, BeyoECL Moon, SDS-PAGE Sample Loading Buffer (5X), TOP2B Rabbit Polyclonal antibody (PROTEINTECH GROUP); Goat Anti-Rabbit IgG, HRP Conjugated (Wuhan Saiweier Biological Technology); pBR322 DNA, DNA Topoisomerase I (Takara Bio); PVDF Membrane(Biotopped); DNA Topoisomerase IIα (TopoGEN). All animal experiments were approved by the guidelines of Animal Ethical and Welfare Committee (#20190036) in Sichuan Agricultural University. And the animals were treated according to the Guide for the Care and Use of Laboratory Animals (Eighth Edition, 2011, The National Academies Press). 2.2 In vitro anti-proliferative activity Taking adriamycin and etoposide as the positive controls, the cytotoxic activities of aconitine linoleate ( 1 ) were measured in vitro on MCF-7 cell line (Breast cancer cell MCF-7) and MCF-7/ADR (adramycin resistant subline) u sing MTS Cell Proliferation Colorimetric Assay Kit (Abdelhaleem et al., 2018 , Luan et al., 2021 ). The cytotoxicity test sample (purity greater than 95%) was dissolved in 1% HCl and adjusted the pH value to 6-7 with 1 mol/L NaOH, then configured into a series of drug concentrations (150, 75, 37.5, 18.75, and 9.375 µg/mL).Take logarithmic growth phase MCF-7 cells, MCF-7/ADR cells, digest and count them with trypsin, prepare a cell suspension with a concentration of 5×10 4 cells/mL with the culture medium, and add 100 µL of cell suspension to each well of a 96-well cell culture plate (Approximately 5×10 3 cells per well). Place the 96-well cell culture plate in a 37 ℃, 5% CO 2 incubator for 24 h. Then culturing these cells for 48 hours with the prepared drugs at various concentrations, MTS solution (20 µL, 2.5 mg/mL in PBS) and 180 µL of culture medium were added for continuous incubation for 2.5h, then measured at 490 nm for the light absorption value. The cell growth inhibition rate was calculated with the equation: Survival fraction = O.D. (treated cells)/O.D. (control cells). The relation between surviving fraction and compound concentration was plotted and IC 50 (the concentration required for 50% inhibition of cell viability) was calculated for each test compound. In order to further explore the selectivity between 1 and normal cells, we tested NIH3T3 cell line (Mouse fibroblast cells) in the same way. (Except that the drug concentration was set to 380, 190, 95, 47.5, 23.75 µg/mL) 2.3 In vitro cell cycle analysis After treating with compound 1 for 48 hours, the MCF-7 cells were washed twice with ice-cold phosphate buffer saline (PBS). The cells was obtained after centrifugation for 5 minutes (1000 r/min), and fixed with 70% ethanol for 6 hours at 4°C, then washed with PBS, re-suspended with 0.1 mg/mL RNase, stained with 40 mg/mL propidium iodide (PI), and analyzed by flow cytometry using FACS Calibur (Becton Dickinson) (Schmitta et al., 2019 ). The cell cycle distributions were calculated using Flowjo.7.6. and SPSS 12.0 software (Becton Dickinson). The interference with the normal cell cycle distribution was indicated after exposure of MCF-7 cells to this compound. 2.4 Topo inhibition assay 2.4.1 Topo I inhibition assay Taking hydroxy camptothecin as positive control, reaction mixtures (20 µL) with supercoile pBR322 DNA (0.25 µg in the 2 µL 10×Tris/Glycine/SDS (TGS) buffer), 1 unit Topo I, 2 µL 0.1% BSA, and 0.2 µL aconitine linoleate ( 1 ) with different concentrations and appropriate distilled deionized water were prepared. After incubating at 37°C for 30 minutes, 10% SDS (1 µL) was added to stop the reaction. A 20 µL aliquot was electrophoresed in 1.2% agarose gel at 80 V for 20 min in 0.5×Tris-acetate (TBE) buffer (20 mM Tris-acetate, 0.5 mM EDTA, pH 8.3), then at 130V for 25 min. After electrophoresis, DNA bands were stained in ethidium bromide (0.5 µg/mL) and visualized by the Bio-Rad gel imaging system. 2.4.2 Topo IIα inhibition assay Taking etoposide as positive control, reaction mixtures (20 µL), containing 0.15 µg of supercoile pBR322 DNA in the 4 µL 10×Tris/Glycine/SDS (TGS) buffer (50 mM spermidine, 720 mM KCl, 350 mM Tris-HCl pH8.0, 50 mM MgCl 2 , 50 mM DTT), 5 units Topo IIα, 0.2 µL aconitine linoleate ( 1 ) with different concentrations and appropriate distilled water, were obtained. After incubating at 37°C for 30 minutes, 10% SDS (1 µL) was added to stop the reaction. An aliquot (20 µL) was electrophoresed in 1.2% agarose gel at 80 V for 20 min in 0.5×Tris-acetate (TBE) buffer (20 mM Tris-acetate, 0.5 mM EDTA, pH 8.3), then at 130V for 25 min. After electrophoresis, DNA bands were stained in ethidium bromide (0.5 µg/mL) and visualized by the Bio-Rad gel imaging system. 2.5 Molecular modeling 2.5.1 Molecular Docking DS BIOVIA Discovery Studio 3.5 and Autodock vina 1.1.2 were used to conduct the molecular docking studies for the binding mode between compound 1 and Topo IIα (Trott and Olson, 2010 ). The three-dimensional (3D) structure of Topo IIα (PDB ID: 5GWK) was obtained from the protein database ( http://www.rcsb.org/pdb/home/home.do ). The 3D structure of the compound was drawn by ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0 software. The docking input files were generated by the AutoDockTools 1.5.6 package (Sanner, 1999 , Morris et al., 2009 ). Ligand structures were prepared for docking by merging non-polar hydrogen atoms and defining rotatable bonds. The search grid of the Topo IIα was identified as center_x: 23.7, center_y: -38.695, and center_z: -60.34 with dimensions size_x: 15, size_y: 15, and size_z: 15. The exhaustive value was set to 20 for improving docking accuracy. For Vina docking, the default parameters were used if it was not mentioned. Then an MD study was performed to revise the docking result. 2.5.2 Molecular Dynamics simulation The AmberTools 15 programs and Amber 14 (Götz et al., 2012 , Pierce et al., 2012 , Salomon-Ferrer et al., 2013 ) were used for MD simulations of the selected docked pose. The compound was first prepared by the tool, ACPYPE (da Silva and Vranken, 2012 ), basing on ANTECHAMBER (Wang et al., 2004 , Wang et al., 2006 ) for generating automatic topologies and parameters in different formats for different molecular mechanics programs, such as calculation of partial charges. Then, the forcefield “leaprc.gaff” (generalized amber forcefield) was used for preparing the ligand, while “leaprc.ff14SB” was used for the receptor. The system was placed in a rectangular box (with a 10.0 Å boundry) of TIP3P water using the “SolvateOct” command with the minimum distance between any solute atoms. PMEMD (Particle Mesh Ewald Molecular Dynamics) module was accelerated by GPU (NVIDIA® Tesla K20c) for a short-term minimization (500 steps per steepest descent and conjugate gradient method), 500 ps heating and 50 ps of density equilibration with weak restraints to reach the equilibrium of solvated complexes. At last, 20 ns of MD simulations were carried out. All the molecular dynamics were performed on Dell Precision T5500 workstation. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method, implemented in AmberTools 15, was used for calculating the the binding free energies (ΔG bind in kcal/mol). Moreover, to identify the key protein residues responsible for the ligands binding process, the binding free energy was decomposed on a per-residue basis. The binding free energy of MM/GBSA for each complex, was estimated by following equations: ΔG bind = G complex ‒G protein ‒G ligand (1) ΔG bind = ΔH‒TΔS ≈ ΔG gas + ΔG sol ‒TΔS (2) ΔG gas = ΔE ele + ΔE vdw (3) ΔG sol = ΔE GB + ΔG SA (4) (where ΔG bind is the binding free energy and G complex , G protein and G ligand are the free energies of complex, protein, and ligand, respectively. The sum of entropy contribution (TΔS) and enthalpy (ΔH) are for the binding free energy. The enthalpy (ΔH) includes the free energy of solvation (ΔG sol ) and free energy of the gas phase (ΔG gas ). ΔG sol is composed of ΔG SA, the nonelectrostatic solvation component (nonpolar contribution) and ΔG GB, the electrostatic solvation energy (polar contribution). ΔG gas includes ΔE ele (electrostatic) and ΔE vdw (van der Waals) energies, which exhibits the gas-phase interaction energy between protein and ligand.) 2.6 Acute toxicity The acute toxicity of the lipo-diterpenoid alkaloids to animal subjects were investigated on the normal mice by using the aconitine linoleate ( 1 ). Briefly, 110 kuming mice (aged 4-5 weeks, weighed 18-22 g, sex in half) were divided randomly into 11 groups, including one negative control group (dilute hydrochloric acid solution, pH 5.4), five dose groups (220, 210, 200, 190, 180 mg/Kg) of aconitine linoleate, and five dose groups (0.6, 0.55, 0.5, 0.4, 0.3 mg/Kg) for the positive control aconitine. All the groups were given the samples by intraperitoneal injection once time. The animals were observed for toxic signs for 4 h after the drug injection. And the changes in physical appearance, injury, pain and signs of illness were recorded daily for the 14 days. Finally, the mice were sacrificed after 14 days and the major tissues, including hart, spleen, kidney, liver, lung and thymus were excised for the analysis of macroscopic pathologicalanatomy. Then the tissues, fixed in formaldehyde 37%, were taken for microscopic evaluation using haematoxylin and eosin staining in the Pathology Laboratory of College of veterinary medicine, Sichuan Agricultural University. The pathological changes of the tissues were assessed and compared. 3. Results 3.1 Biology Taking adriamycin and etoposide as positive control, the in vitro anti-proliferative activity of Aconitine linoleate ( 1 ) was examined against MCF-7 and MCF-7/ADR cell lines by MTS method (Ahmed et al., 2017). The results were expressed as IC 50 (median growth inhibitory concentration) values, representing the concentrations needed to produce a 50% inhibition of cell growth after incubation for 48 h (Table 1 and Figure 2) (Luan et al., 2021). In order to further explore the selectivity between 1 and normal cells, we used NIH3T3 cell line as normal cells for comparison and found that the IC 50 was 72.08±0.15 μM, and selective index (SI)=9.51 and 10.26 respectively compared with MCF-7 and MCF-7/ADR (Table 1). To explore the effect of the Aconitine linoleate on cell proliferation of MCF-7/ADR cells, we analyzed cell cycle distribution of aconitine linoleate ( 1 ) at its IC 50 concentration (Table 1) using flow cytometry assay. Our data in Figure 3 shows that aconitine linoleate ( 1 ), containing 8-linoleic acid side chain, 3-OH and N-ethyl group, exhibits the potent anti-proliferative effect on MCF-7/ADR cells by increasing percentage of cells G 0 G 1 phase, decreasing the same in S phase. 3.2 Molecular docking To better understand the potential binding targets of lipo-diterpenoid alkaloids, the molecular docking of aconitine linoleate ( 1 ) with 18 kinds of common antitumor targets was first screened by Discovery Studio 3.5. The docking results in Table 2, indicated the most favorable target would be Topo II for the highest Libdockscore (324.932). Interestingly, Topo IIα may be the selective target (Libdockscore of 102.535), rather than Topo IIβ, which has been failed in docking process. In contrast, aconitine ( 4 ) could not successfully dock with those two targets (Table 3), relating to the bad anti-proliferative activity of aconitine against those two cancer cells. The potential binding mode between compound 1 and Topo IIα has been further explored by molecular docking and molecular dynamic simulations, using the AutoDock vina 1.1.2 and Amber14 software. Bonding models of 1 and Topo IIα were depicted in Figure 4, showing the residue A/Arg-487 close to the phenyl group of the compound 1 by cation-π interaction (Figure 4A). To gain more information about the residues surrounding the binding site and their contribution to the whole system, the MMGBSA approach was used to calculate the contribution of the electrostatic, solvation, Van der Waals force, and residues to the free energy of binding. Each residue interaction free energy was presented as electrostatic (∆ E ele ), solvation (∆ E sol ), Van der Waals (∆ E vdw ) and total contribution (∆ E total ). In the Topo IIα- 1 complex, the residue A/Arg-487 has a strong electrostatic (∆ E ele ) contribution, with the ∆ E ele of < -11.0 kcal/mol (Figure 4B). In addition, due to the close proximity between the nucleotides and the compound 1 , strong Van der Waals interactions (< -4.5 kcal/mol) between nucleotides C/DC-8, D/DT-9, F/DA-12 and F/DG-13, and the ligand have been detected. Overall, the majority of the decomposed energy interaction originated from Van der Waals interactions, mainly through hydrophobic interactions, such as A/Met-762, A/Met-766 and B/Ala-801. It referred that the introduction of hydrophobic groups, such as halogen, nitro, benzene ring, etc., into the molecule, may increase the binding force with Topo IIα, thereby enhancing its anti-proliferative activity. Furthermore, the calculation of the total binding free energy for the Topo IIα- 1 complex led to an estimated ∆G bind of -9.1 kcal/mol for 1 (Table 4), indicating the strongly bind of 1 to the binding site of the Topo IIα. This suggests that the anti-proliferative activity of 1 is very likely to take effect through the complex with Topo IIα. The docking results of 20-ns molecular dynamics simulation confirmed the preferential binding mechanism of Topo IIα and 1 . The root mean square deviation (RMSD) values of the protein skeleton basing on the initial structure with the simulation time was performed to figure out the dynamic stability of the models and ensure the rationality of the sampling strategy (Figure 4C), which proved the stability of protein in two systems under the simulation. The flexibility of the whole protein residues in Topo IIα-1 complex and free Topo IIα were detected by the root mean square fluctuations (RMSF) values (Figure 4D), presenting the difference in flexibility between the binding site of Topo IIα with 1 . The results presented a small degree of flexibility with a RMSF of less than 3 Å at the binding site, referring these residues seem to be more rigid as a result of binding to 1 . In summary, the rational explanation of the interactions between 1 and Topo IIα by the molecular dynamic simulations provided valuable information for further development of the Topo IIα inhibitors. In order to prove the molecular docking results, the activity inhibition of topoisomerase enzyme with compound 1 was suspected as expecting mechanism for the action of lipo-diterpenoid alkaloids.The assay was performed to evaluate the Topo I, IIα inhibitory activity (Figure 5). Aconitine linoleate ( 1 ) exhibited promising inhibition activity below 200 μM, comparable to the known potent Topo IIα inhibitor, etoposide. Whereas, it exhibited no obvious inhibition against Topo I, even at the concentration of 200 μM. Thus, it was proved that compound 1 exerted its anti-proliferative activity through selective Topo IIα inhibition, basing the results of both molecular docking and inhibition of topoisomerase activity. However, in the Topo IIα inhibition assay, the IC 50 values were much higher than in the cytotoxicity assays. It can be concluded that compound 1 possessed the anti-proliferative activity partially dependent on the Topo IIα inhibition. 3.3 Acute toxicity For further application, the toxicity evaluation should be explored as an important factor. To investigate the toxicity of lipo-diterpenoid alkaloids, the acute toxicity test of aconitine linoleate ( 1 ) was carried out in Kuming mice for the LD 50 (half lethal dose) value. The results showed that the LD 50 value of aconitine linoleate ( 1 ) was 2.2×10 5 nmol/Kg. Compared with aconitine (LD 50 =4.5×10 2 nmol/Kg), the safe dosage of aconitine linoleate to mice is 489 times higher than aconitine, suggesting the obvious toxicity reduction for lipo-diterpenoids (Table 5). Furthermore, as shown in Figure 6, after aconitine linoleate treatment, no obvious pathological lesions appear in the main tissues, including hart, spleen, kidney, liver, lung and thymus. The pathological morphology in mice tissue with/without compound 1 (2.2×10 5 nmol/Kg) or aconitine (4.5×10 2 nmol/Kg) were also conducted by H&E staining method. Compared with the blank group, only liver and lung tissue in the aconitine linoleate group showed slight pathological changes, which are much milder than the aconitine group. For liver tissue, the obvious damage is found in the aconitine group with moderate diffuse vacuolar degeneration and punctate necrosis foci, which are occasionally seen in the aconitine linoleate group. For lung tissue, the alveolar septum is significantly thicker and the number of alveoli is decreased in the aconitine group; whereas, the alveolar septum in the aconitine linoleate group is slightly thicker, significantly better than that of the aconitine group. 4. Discussion Firstly, compared with aconitine (4), aconitine linoleate (1) exhibited high in vitro cytotoxic activities against both MCF-7 and MCF-7/ADR cell lines, and displayed much lower toxicity in the in vivo acute toxicity evaluation in mice. Based on the results of molecular docking, aconitine linoleate (1) selectively well docked with Topo IIα, while aconitine (4) failed. Considering the tumor targeting effect of long chain fatty acids (Sauer et al., 2000), it is concluded that the introduction of 8-lipo group significantly enhanced its affinity and selectivity for Topo IIα, as well as increasing the tumor targeting which led to the low toxicity. Secondly, according to the data of flow cytometry assay, aconitine linoleate ( 1 ) exhibits cytotoxic effect on MCF-7/ADR cells by increasing percentage of cells G0/G1 phase, decreasing the same in S phase. As everyone knows, Topo IIα and IIβ are two types of Topo II in humans. The expression level of Topo IIα is the highest in G2/M phase, followed by S phase, and lowest in G0/G1 phase, but that of Topo IIβ is always constant (Jun et al., 2015). Therefore, due the selective inhibition of Topo IIα, aconitine linoleate ( 1 ) targeted the tumor cells in S phase. Thirdly, as a widely used chemotherapeutic agent, doxorubicin, mainly targeting Topo IIα (Du et al., 2011), has caused drug-resistance during clinical application, which is a complex process (Ganapath and Ganapathi, 2013). Different resistant mechanisms have been recognized, including the enhanced expression of the P-glycoprotein, ABCB1, ABCG2, the down-expression level of topo IIα, overexpression of multidrug resistance protein 1(MDR1), and even the activation of autophagy (Mechetner et al., 1998, Latorre et al., 2012, Shatha AbuHammad and Zihlif, 2013, Guo et al., 2016). Although targeted to the same enzyme, aconitine linoleate (1) is mainly a topo catalytic inhibitor, which could selectively inhibit Topo IIα, rather than a topo poison like doxorubicin. They may have different topo-inhibit mechanism. Moreover, the reversal mechanism of tumor drug resistance of aconitine linoleate (1) should be a multi-targeted way, which need further explore in the next step. 5. Conclusions We conclude that aconitine linoleate ( 1 ) not only selectively inhibits the expression and activity of Topo IIα, but also induce a significant increase in the percentage of cells at G 0 G 1 phase, leading its excellent anti-proliferative activity against MCF-7 and MCF-7/ADR in vitro . Since the introduction of 8-lipo group, 1 is much less toxic than aconitine. Lipo-alkaloid would be a powerful and potentially anti-tumor compound against drug-resistant breast cancer, bringing more possibilities for fighting breast cancer in the future. Associated Content Supporting Information The Supporting Information is available free of charge on website at DOI: Data availability As supplemental material and upon request. Accession Codes The PDB access code for structure of 1 bond to topoisomerase IIα is 5GWK. Authors will release the atomic coordinates and experimental data upon article publication. Declarations Competing interests 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. Ethics approval and consent to participate The animal use protocol listed below has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee. Consent for publication All authors approved the manuscript to be published. Author contributions Shangxian Luan: Writing paper and participating in Molecular Docking and in vitro anti-proliferative activity, acute toxicity. Yingying Gao: Participating in in vitro cell cycle analysis and Topo inhibition assay. Xiaoxia Liang: Designing and leading experiments, writing and revising the paper. Li Zhang: participating in acute toxicity. Qiang Wu: participating in acute toxicity. Yunkai Hu: participating in acute toxicity. Shixi Liu: participating in Molecular Docking. Lizi Yin and Changliang He guided the experiment. The authors declare that all data were generated in-house and that no paper mill was used. Funding This study was supported by the National Natural Science Foundation of China Youth Fund and the National Natural Science Foundation of China (Grant No. 81703387, 31972743) References Abdelhaleem EF, Abdelhameid MK, Kassab AE, Kandeel MM (2018) Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity against breast cancer cell line MCF-7. 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Journal of Natural Medicines 74: 83-89. https://doi.org/10.1007/s11418-019-01346-z Tables Table 1 The IC 50 (μM) results of compounds 1 and 4 against MCF-7, MCF-7/ADR and NIH3T3 No. MCF-7 MCF-7/ADR NIH3T3 1 7.58±0.06 a,b 7.02±0.12 a,b 72.08±0.15 4 >400 86.35±0.32 a,b, c - Adriamycin 1.22±0.43 a,b,c 67.61±6.50 b, c - Etoposide 18.01±1.64 a,b,c 35.48±0.29 a, c - SI 9.51 - 10.26 Note: Compared with positive control group ( a Adriamycin, b Etoposide) a P < 0.05, b P < 0.05; Compared with 1 group, c P < 0.05; - means no data, SI: selectivity of compound 1 between MCF-7 or MCF-7/ADR and NIH3T3 cells . Table 2 Docking results of aconitine linoleate ( 1) and receptor protein molecules Receptor protein PDB Number Libdockscore Poses Topo Ⅱ 5bTc 324.932 26 Topo Ⅰ 1nh3 83.3349 1 Epidermal Growth Factor HER2 3WSQ 147 23 Epidermal Growth Factor HER3 5O4O 142 93 Matrix metalloproteinases 1bqo 109.144 1 human basic fibroblast growth factor 1bfb 0 0 Human vascular endothelial growth factor 1vpf 0 0 Protein kinase C 2kf9 0 0 Insulin receptor 3ekk 0 0 Platelet-derived growth factor receptor 3mjg 144.6 56 Cytochrome P450 3A4 4i3q 133.45 4 Angiogenin 1 4k0v 138.278 16 Angiogenin 2 4iml 132.827 32 Human tyrosine kinase c-KIT 6gqj 121.48 7 Human cyclooxygenase Ⅱ 5f19 0 0 Epidermal growth factor receptor 1ivo 120 22 Note: LibDockscore indicates the degree of docking between receptors and ligands; Poses represents the way ligands bind to proteins; If the score and poses are 0, the docking failed. Table 3 Comparation of the docking results of between aconitine linoleate ( 1 ) and aconitine (4) with receptor protein Topo Ⅱα and Topo Ⅱβ Compound Receptor protein PDB Number Libdockscore Poses 1 Topo Ⅱα 4fm9 102.535 13 Topo Ⅱβ 4g0w 0 0 4 Topo Ⅱα 4fm9 0 0 Topo Ⅱβ 4g0w 0 0 Note: LibDockscore indicates the degree of docking between receptors and ligands; Poses represents the way ligands bind to proteins; If the score and poses are 0, the docking failed. Table 4. Average binding free energies (kcal mol −1 ) for the Topo2α-1 complex along with the different energy contributions Compd ΔEvdw ΔEele ΔGgas ΔEGB ΔGSA ΔGsol ΔH TΔS ΔGbind L29 -99.8 -45.6 -145.4 88.7 -11.0 77.7 -67.7 -58.6 -9.1 Table 5. Acute toxicity test results of compound 1 and 4 in mi ce through celiac injection Compound Concentration (mg/Kg) Number of deaths LD 50 (nmol/Kg) 1 180 3 190 4 200 6 210 7 220 9 2.2×10 5 4 0.3 1 0.4 2 0.5 3 0.55 6 0.6 9 4.5×10 2 Note: A total of 11 groups including the negative control group (dilute hydrochloric acid solution, pH 5.4), each with 10 mice; There was no death in the negative control group. Supplementary Files MCF7Compound1.xls MCF7adriamycin.xls MCF7etoposide.xls MCF7ADRCompound1.xls MCF7ADRadriamycin.xls MCF7ADRetoposide.xls NIH3T3Compound1.xls SIJE.doc Onlinefloatimage1.png Graphical Abstract Cite Share Download PDF Status: Under Review Version 1 posted First submitted to journal 09 Oct, 2021 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-1005235","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":58282464,"identity":"3d4ce744-0669-43b3-82c9-d0602bb1e08f","order_by":0,"name":"Shangxian Luan","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shangxian","middleName":"","lastName":"Luan","suffix":""},{"id":58282465,"identity":"eeaaa45a-7549-44ef-bf5b-22df9bb99fe3","order_by":1,"name":"Yingying Gao","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Gao","suffix":""},{"id":58282466,"identity":"693f629b-3022-4df3-a8a1-52060e8fc524","order_by":2,"name":"Xiaoxia Liang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBACPmYwJQHEjA0MH8AMIODBo4UNWQvjDKK0IHOY4SrxamFnfvbwa5uFPL9EcuNnmz9AxowExgdv2xjkzXE6jM3cWOaMhOHMnoPN0rltEoYzbiQwG85tYzDc2YDTL2bSEhUSjBuONzZI5zZIJDDcSGCT5m1jSDA4gEsL+zdpCQMJ+w2HGZt/W/yRSJC/kcD+G78WHjPJDxUSiUBb2qQZ2CQSDIC2MBPQUibNcEYiGeiXNsteoF82nnnYLDnnnIThBhxa+PmPb5P82VZn2y+R/vjGjz918nLHkw9+eFNmI4/LFhBgRosFYDJggMUpDsD4A6/0KBgFo2AUjHgAABOUUDwnJNpXAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5002-5327","institution":"Sichuan Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Liang","suffix":""},{"id":58282467,"identity":"20f357d8-5a8a-43de-9d95-fade6d78d2a6","order_by":3,"name":"Li Zhang","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhang","suffix":""},{"id":58282468,"identity":"2111b352-3180-496e-a404-b99b183b0bb6","order_by":4,"name":"Qiang Wu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wu","suffix":""},{"id":58282469,"identity":"209af3e3-fec2-487b-9a6d-8a57936df7c9","order_by":5,"name":"Yunkai Hu","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunkai","middleName":"","lastName":"Hu","suffix":""},{"id":58282470,"identity":"7a3c87da-acf8-47f3-ac35-de5ca6323b9b","order_by":6,"name":"Lizi Yin","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lizi","middleName":"","lastName":"Yin","suffix":""},{"id":58282471,"identity":"8921c15b-37f5-4ced-95a9-2b5f82ebb722","order_by":7,"name":"Changliang He","email":"","orcid":"","institution":"Sichuan Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Changliang","middleName":"","lastName":"He","suffix":""},{"id":58282472,"identity":"e3aa2192-940a-4018-bb2f-0357286d5061","order_by":8,"name":"Shixi Liu","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shixi","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2021-10-21 19:12:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1005235/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1005235/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":14796730,"identity":"b8028e9c-752a-44ed-a116-f764258e033b","added_by":"auto","created_at":"2021-10-22 14:02:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29823,"visible":true,"origin":"","legend":"Part of the reported studies of C19-diterpenoid alkaloids with cytotoxicity","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/3e8d3402f1f3e8abe8d294f3.png"},{"id":14796335,"identity":"d1289aa9-109b-4015-8846-7b75a0b4d76e","added_by":"auto","created_at":"2021-10-22 13:56:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20061,"visible":true,"origin":"","legend":"The in vitro anti-proliferative activity of Aconitine linoleate (1), adriamycin and etoposide examined against MCF-7 (Breast Cancer) and MCF-7/ADR cell lines. (Low: 5 μM, Medium: 10 μM, High: 15 μM)","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/83b9643f2e6c1fc4a5c42ca5.png"},{"id":14795840,"identity":"6fffe4d2-9a0e-448b-b23e-26592487305d","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148602,"visible":true,"origin":"","legend":"Effect of aconitine linoleate (1) on the proliferation cycle of MCF-7/ADR cell","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/42c0c905945a7281d90a9236.png"},{"id":14796114,"identity":"15509d7a-311e-4825-add8-03dd8866528c","added_by":"auto","created_at":"2021-10-22 13:53:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":281811,"visible":true,"origin":"","legend":"Topo Ⅱα protein molecules. A) The predicted binding mode of 1 in Topo IIα binding pocket obtained from MD simulation. B) Decomposition of the binding energy on a per-residue basis in the Topo IIα-1 complex. C) The root-mean-square deviations (RMSDs) of all the atoms of Topo IIα-1 complex with respect to its initial structure as function of time. D) RMSF of residues of the whole protein in Topo IIα-1 complex and free Topo IIα during the 20 ns simulation. ","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/981cab0f8b0db91dad239de6.png"},{"id":14795846,"identity":"930b5ef2-e94f-481e-81b9-d412f0c68adb","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44188,"visible":true,"origin":"","legend":"The Topoisomerase activity inhibition of aconitine linoleate (1). (a) Aconitine linoleate (1) inhibited the Topo Ⅰ mediated pBR322 DNA relaxation; (b) Aconitine linoleate (1) inhibited the Topo Ⅱα mediated pBR322 DNA relaxation.","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/24054d33db7ffa18a156a02e.png"},{"id":14795848,"identity":"cd779302-e9a7-46a3-86d8-8a4cfc5aa8bc","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":387904,"visible":true,"origin":"","legend":"The acute toxicity of aconitine linoleate (1) on mice. A) Histological analysis of the liver and lung tissues from acute toxicity test model (blank control group; 180 mg/kg for aconitine linoleate group; 0.2 mg/kg for aconitine group). Photomicrograph of tissue stained with H\u0026E staining, ×400. B-D). The vertical arrows on liver figures indicate vacuolar degeneration and the horizontal arrow indicates necrosis; the arrows on lung figures indicate the alveolar septum thickening. B) Acute toxicity test with 1 (180 mg/Kg group) in mice through celiac injection, n= 10. C) The main tissue of aconitine linoleate (1) in different concentration (180, 190, 200, 210, 220 mg/Kg) and control group. D) The mice carcass after 14 d (180 mg/Kg group). (Note: The vertical arrows in A indicate vacuolar degeneration and the horizontal arrow indicates necrosis in liver and the alveolar septum thickening in lung, respectively.)","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/88f82d5b2ceae3e3796eec2c.png"},{"id":14796731,"identity":"25cb24a3-8916-4da6-9cd7-584108ec880e","added_by":"auto","created_at":"2021-10-22 14:02:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1327338,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/1675aadd-c22e-4dfb-8308-1ffa4b6269fe.pdf"},{"id":14796113,"identity":"3f4d524f-692d-457f-bca9-58b256cfda6b","added_by":"auto","created_at":"2021-10-22 13:53:31","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":42496,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7Compound1.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/d8b46b60203ab2f84e165d74.xls"},{"id":14795838,"identity":"1b5849af-1bc7-44e2-a94f-f96f5cdaf141","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"xls","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35840,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7adriamycin.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/b5d02c76f7c0fdba2b64a98f.xls"},{"id":14796116,"identity":"726107a8-3efc-47d4-a1d6-8e9047997b5e","added_by":"auto","created_at":"2021-10-22 13:53:31","extension":"xls","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":41472,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7etoposide.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/34c56442044a47a7a959006d.xls"},{"id":14796333,"identity":"540903f9-1590-4179-a2a0-723d068d708e","added_by":"auto","created_at":"2021-10-22 13:56:31","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16384,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7ADRCompound1.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/122d29045e7b20a1f8976434.xls"},{"id":14796569,"identity":"f56257e6-048c-4672-9a29-7014dde2030e","added_by":"auto","created_at":"2021-10-22 13:59:31","extension":"xls","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16384,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7ADRadriamycin.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/f550449dee0285fc8db791d2.xls"},{"id":14795852,"identity":"6c280b9d-e227-41c0-a319-7bd193831db5","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":35840,"visible":true,"origin":"","legend":"","description":"","filename":"MCF7ADRetoposide.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/9367e4e4f27f7c3d22c90004.xls"},{"id":14796337,"identity":"df5c17e5-5e3e-4bda-a979-fdc8ccbc08bf","added_by":"auto","created_at":"2021-10-22 13:56:31","extension":"xls","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":33792,"visible":true,"origin":"","legend":"","description":"","filename":"NIH3T3Compound1.xls","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/7c44beccc5a16efe9287c83f.xls"},{"id":14795851,"identity":"4a13b2e8-087c-427d-adf4-811f8f92fc3e","added_by":"auto","created_at":"2021-10-22 13:50:31","extension":"doc","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2733056,"visible":true,"origin":"","legend":"","description":"","filename":"SIJE.doc","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/f426525f2f71b37b3e518de8.doc"},{"id":14796120,"identity":"bcc79941-3682-4538-bb05-4f79f0d8ef95","added_by":"auto","created_at":"2021-10-22 13:53:31","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":54192,"visible":true,"origin":"","legend":"Graphical Abstract","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1005235/v1/363e58af585442112c9973a8.png"}],"financialInterests":"","formattedTitle":"Aconitine linoleate, a natural lipo-diterpenoid alkaloid, stimulates anti-proliferative activity reversing doxorubicin-resistant in MCF-7/ADR breast cancer cells as a selective topoisomerase IIα inhibitor","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAccording to the statistical data from the World Health Organization (WHO) in 2020, breast cancer has taken the top one place both in diagnose rate and mortality rate for female (Sung et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, its treatment has been hindered by multidrug resistance (MDR), leading to the urge for new antitumor agent targeted specific target, especially for reversing the resistant problems. Among the various anticancer targets, DNA topoisomerases (topos) are well-known ones (Li and Liu, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, Pommier, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, McClendon and Osheroff, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Nitiss, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2009b\u003c/span\u003e, Nitiss, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e). Topos inhibitors are generally classified into Topo I inhibitors as camptothecin (CPT) (Schoeffler and Berger, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and Topo II inhibitors as amsacrine, etoposide and doxorubicin (Khadka and Cho, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). However, the clinic use of current anticancer topoisomerase inhibitors have been limited by some major negative consequences. Firstly, most clinically active topo inhibitors are topo poisons, such as etoposide and doxorubicin, which usually need monitoring for the severe genetic toxicity (Jun et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Secondly, the severe dose-limiting toxicities and drug resistance of Topo I inhibitors like CPT have been emerged. Thirdly, the secondary malignancies led by the inhibition of Topo IIβ in the clinic use of the unselective Topo II inhibitors have been reported (Felix, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Mistry et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Azarova et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). For these reasons, many researchers have targeted Topo IIα, rather than IIβ, on developing topo catalytic inhibitors, which induces cancer cell death through the elimination of the essential enzymatic activity, rather than topo poisons(Albert et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs a widely used traditional Chinese medicine, \u003cem\u003eAconitum\u003c/em\u003e possesses anti-inflammatory, analgesic, and anti-cancer activities (Kim et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2002\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). Single \u003cem\u003eAconitum\u003c/em\u003e and its prescriptions have been documented for the treatment of cancer since last century in China (Mingxin Tang and Sun, 1986, He Shu et al., 2009, Chen, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Qiuping Xu et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Duan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the precise mechanisms of the \u003cem\u003eAconitum\u003c/em\u003e eliciting anticancer effects have not yet been fully clarified due to the diverse structures of compounds in \u003cem\u003eAconitum\u003c/em\u003e. Diterpenoid alkaloids, the main active components from \u003cem\u003eAconitum\u003c/em\u003e, have been reported as potential cytotoxic agents for decades (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e, Hazawa et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e, Hazawa et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009b\u003c/span\u003e, Wada et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Wada, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wada et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Wada et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, Wada et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Wada K., 2019) (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). But the significant toxicities of diterpenoid alkaloids, such as the neurotoxicity and cardiotoxicity of aconitine (\u003cb\u003e4\u003c/b\u003e), limited their further clinical application. Lipo-diterpenoid alkaloids, a kind of diterpenoid alkaloids bearing long ester groups (such as \u003cb\u003e1-3\u003c/b\u003e in Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), are common components obtained from \u003cem\u003eAconitum\u003c/em\u003e, which have been reported with extensive pharmacological activities and low toxicity (Kitagawa et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1982\u003c/span\u003e, Isao et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1984\u003c/span\u003e, Hanuman and Katz, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e, Yamashita et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Yamashita et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In 2017, our group first reported the obvious cytotoxic activities of lipo-diterpenoids \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e, against HL-60, A-549, SMCC-7721, MCF-7 and SW480 cell lines, comparable to cisplatin (Liang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e, Liang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Later, the good cytotoxic activities of one lipo-alkaloid against several tumor cell lines (A549, MDA-MB-231, MCF-7, KB, KB-VIN) also have been reported by Koji Wada group (Koji Wada, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Yamashita H., 2020). Then, the highly selective index (SI \u0026gt;10) for compound \u003cb\u003e1\u003c/b\u003e between MCF-7 and mouse fibroblast cells, has surprised us, arousing a deeper pharmacological mechanism research in current study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this work, aconitine linoleate (\u003cb\u003e1\u003c/b\u003e) was investigated for its anti-proliferative activity \u003cem\u003ein vitro\u003c/em\u003e against MCF-7 and adramycin resistant subline MCF-7/ADR cell lines. In order to explore its mechanistic pathways, the lead compound \u003cb\u003e1\u003c/b\u003e was performed extra investigations such as cell cycle analysis, topoisomerase I / II inhibition, and molecular docking. Meanwhile, for determining its safety the acute toxicity on mice was also tested.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e \u003cp\u003eAconitine linoleate (\u003cb\u003e1\u003c/b\u003e) (isolated from the \u003cem\u003eAconitum sinchiangense\u003c/em\u003e W. T. Wang, plants were collected from Yili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China, in 2016, and was identified by Dr. Lixia Li. The plants are Stored in the Natural Medicine Research Center of Sichuan Agricultural University, the voucher specimen No. 20140616-1); MCF-7 cell line (Breast cancer cell MCF-7), MCF-7/ADR (adramycin resistant subline) and NIH3T3 cell line (Mouse fibroblast cells) (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences); Kuming mice (CHENGDU DOSSY EXPERIMENTAL ANIMALS), PBS (HyClone); DMEM high glucose (HyClone); fetal bovineserum (FBS) (Gibco); propidium iodide (PI) (Yeasen Biotech); Adriamycin (RYON); Etoposide and hydroxy camptothecin (TargetMol);Trypsin-EDTA (0.25%), phenol red (solarbio); Penicillin-Streptomycin Liquid (solarbio); CellTiter 96\u0026reg;AQueous One Solution Cell Proliferation Assay (Promega); Paraffin wax (Shanghai Hualing kangfu Machinery Factory); Ehrlich hematoxylin stain and Eosin Solution (solarbio); LDH Cytotoxicity Assay Kit (solarbio); Prestained Protein Ladder, 10 to 180 kDa and 10-170 kDa, RIPA Lysis Buffer, Topoisomerase Ⅱ alpha Rabbit Monoclonal Antibody, Topoisomerase Ⅰ Rabbit Monoclonal Antibody, BeyoECL Moon, SDS-PAGE Sample Loading Buffer (5X), TOP2B Rabbit Polyclonal antibody (PROTEINTECH GROUP); Goat Anti-Rabbit IgG, HRP Conjugated (Wuhan Saiweier Biological Technology); pBR322 DNA, DNA Topoisomerase I (Takara Bio); PVDF Membrane(Biotopped); DNA Topoisomerase IIα (TopoGEN).\u003c/p\u003e \u003cp\u003e All animal experiments were approved by the guidelines of Animal Ethical and Welfare Committee (#20190036) in Sichuan Agricultural University. And the animals were treated according to the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (Eighth Edition, 2011, The National Academies Press).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 In vitro anti-proliferative activity\u003c/h2\u003e \u003cp\u003eTaking adriamycin and etoposide as the positive controls, the cytotoxic activities of aconitine linoleate (\u003cb\u003e1\u003c/b\u003e) were measured \u003cem\u003ein vitro\u003c/em\u003e on MCF-7 cell line (Breast cancer cell MCF-7) and MCF-7/ADR (adramycin resistant subline) \u003cem\u003eu\u003c/em\u003esing MTS Cell Proliferation Colorimetric Assay Kit (Abdelhaleem et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Luan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The cytotoxicity test sample (purity greater than 95%) was dissolved in 1% HCl and adjusted the pH value to 6-7 with 1 mol/L NaOH, then configured into a series of drug concentrations (150, 75, 37.5, 18.75, and 9.375 \u0026micro;g/mL).Take logarithmic growth phase MCF-7 cells, MCF-7/ADR cells, digest and count them with trypsin, prepare a cell suspension with a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL with the culture medium, and add 100 \u0026micro;L of cell suspension to each well of a 96-well cell culture plate (Approximately 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well). Place the 96-well cell culture plate in a 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h. Then culturing these cells for 48 hours with the prepared drugs at various concentrations, MTS solution (20 \u0026micro;L, 2.5 mg/mL in PBS) and 180 \u0026micro;L of culture medium were added for continuous incubation for 2.5h, then measured at 490 nm for the light absorption value. The cell growth inhibition rate was calculated with the equation: Survival fraction = O.D. (treated cells)/O.D. (control cells). The relation between surviving fraction and compound concentration was plotted and IC\u003csub\u003e50\u003c/sub\u003e (the concentration required for 50% inhibition of cell viability) was calculated for each test compound. In order to further explore the selectivity between \u003cb\u003e1\u003c/b\u003e and normal cells, we tested NIH3T3 cell line (Mouse fibroblast cells) in the same way. (Except that the drug concentration was set to 380, 190, 95, 47.5, 23.75 \u0026micro;g/mL)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 In vitro cell cycle analysis\u003c/h2\u003e \u003cp\u003eAfter treating with compound \u003cb\u003e1\u003c/b\u003e for 48 hours, the MCF-7 cells were washed twice with ice-cold phosphate buffer saline (PBS). The cells was obtained after centrifugation for 5 minutes (1000 r/min), and fixed with 70% ethanol for 6 hours at 4\u0026deg;C, then washed with PBS, re-suspended with 0.1 mg/mL RNase, stained with 40 mg/mL propidium iodide (PI), and analyzed by flow cytometry using FACS Calibur (Becton Dickinson) (Schmitta et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The cell cycle distributions were calculated using Flowjo.7.6. and SPSS 12.0 software (Becton Dickinson). The interference with the normal cell cycle distribution was indicated after exposure of MCF-7 cells to this compound.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.4\u003c/em\u003e Topo \u003cem\u003einhibition assay\u003c/em\u003e\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Topo I inhibition assay\u003c/h2\u003e \u003cp\u003eTaking hydroxy camptothecin as positive control, reaction mixtures (20 \u0026micro;L) with supercoile pBR322 DNA (0.25 \u0026micro;g in the 2 \u0026micro;L 10\u0026times;Tris/Glycine/SDS (TGS) buffer), 1 unit Topo I, 2 \u0026micro;L 0.1% BSA, and 0.2 \u0026micro;L aconitine linoleate (\u003cb\u003e1\u003c/b\u003e) with different concentrations and appropriate distilled deionized water were prepared. After incubating at 37\u0026deg;C for 30 minutes, 10% SDS (1 \u0026micro;L) was added to stop the reaction. A 20 \u0026micro;L aliquot was electrophoresed in 1.2% agarose gel at 80 V for 20 min in 0.5\u0026times;Tris-acetate (TBE) buffer (20 mM Tris-acetate, 0.5 mM EDTA, pH 8.3), then at 130V for 25 min. After electrophoresis, DNA bands were stained in ethidium bromide (0.5 \u0026micro;g/mL) and visualized by the Bio-Rad gel imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Topo IIα inhibition assay\u003c/h2\u003e \u003cp\u003eTaking etoposide as positive control, reaction mixtures (20 \u0026micro;L), containing 0.15 \u0026micro;g of supercoile pBR322 DNA in the 4 \u0026micro;L 10\u0026times;Tris/Glycine/SDS (TGS) buffer (50 mM spermidine, 720 mM KCl, 350 mM Tris-HCl pH8.0, 50 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 50 mM DTT), 5 units Topo IIα, 0.2 \u0026micro;L aconitine linoleate (\u003cb\u003e1\u003c/b\u003e) with different concentrations and appropriate distilled water, were obtained. After incubating at 37\u0026deg;C for 30 minutes, 10% SDS (1 \u0026micro;L) was added to stop the reaction. An aliquot (20 \u0026micro;L) was electrophoresed in 1.2% agarose gel at 80 V for 20 min in 0.5\u0026times;Tris-acetate (TBE) buffer (20 mM Tris-acetate, 0.5 mM EDTA, pH 8.3), then at 130V for 25 min. After electrophoresis, DNA bands were stained in ethidium bromide (0.5 \u0026micro;g/mL) and visualized by the Bio-Rad gel imaging system.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Molecular modeling\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Molecular Docking\u003c/h2\u003e \u003cp\u003eDS BIOVIA Discovery Studio 3.5 and Autodock vina 1.1.2 were used to conduct the molecular docking studies for the binding mode between compound \u003cb\u003e1\u003c/b\u003e and Topo IIα (Trott and Olson, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The three-dimensional (3D) structure of Topo IIα (PDB ID: 5GWK) was obtained from the protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.rcsb.org/pdb/home/home.do\u003c/span\u003e\u003c/span\u003e). The 3D structure of the compound was drawn by ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0 software. The docking input files were generated by the AutoDockTools 1.5.6 package (Sanner, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, Morris et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Ligand structures were prepared for docking by merging non-polar hydrogen atoms and defining rotatable bonds. The search grid of the Topo IIα was identified as center_x: 23.7, center_y: -38.695, and center_z: -60.34 with dimensions size_x: 15, size_y: 15, and size_z: 15. The exhaustive value was set to 20 for improving docking accuracy. For Vina docking, the default parameters were used if it was not mentioned. Then an MD study was performed to revise the docking result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2 Molecular Dynamics simulation\u003c/h2\u003e \u003cp\u003eThe AmberTools 15 programs and Amber 14 (G\u0026ouml;tz et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Pierce et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Salomon-Ferrer et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) were used for MD simulations of the selected docked pose. The compound was first prepared by the tool, ACPYPE (da Silva and Vranken, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), basing on ANTECHAMBER (Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) for generating automatic topologies and parameters in different formats for different molecular mechanics programs, such as calculation of partial charges. Then, the forcefield \u0026ldquo;leaprc.gaff\u0026rdquo; (generalized amber forcefield) was used for preparing the ligand, while \u0026ldquo;leaprc.ff14SB\u0026rdquo; was used for the receptor. The system was placed in a rectangular box (with a 10.0 \u0026Aring; boundry) of TIP3P water using the \u0026ldquo;SolvateOct\u0026rdquo; command with the minimum distance between any solute atoms. PMEMD (Particle Mesh Ewald Molecular Dynamics) module was accelerated by GPU (NVIDIA\u0026reg; Tesla K20c) for a short-term minimization (500 steps per steepest descent and conjugate gradient method), 500 ps heating and 50 ps of density equilibration with weak restraints to reach the equilibrium of solvated complexes. At last, 20 ns of MD simulations were carried out. All the molecular dynamics were performed on Dell Precision T5500 workstation. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method, implemented in AmberTools 15, was used for calculating the the binding free energies (ΔG\u003csub\u003ebind\u003c/sub\u003e in kcal/mol). Moreover, to identify the key protein residues responsible for the ligands binding process, the binding free energy was decomposed on a per-residue basis. The binding free energy of MM/GBSA for each complex, was estimated by following equations: ΔG\u003csub\u003ebind\u003c/sub\u003e= G\u003csub\u003ecomplex\u003c/sub\u003e‒G\u003csub\u003eprotein\u003c/sub\u003e‒G\u003csub\u003eligand\u003c/sub\u003e (1) ΔG\u003csub\u003ebind\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔH‒TΔS\u0026thinsp;\u0026asymp;\u0026thinsp;ΔG\u003csub\u003egas\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔG\u003csub\u003esol\u003c/sub\u003e‒TΔS (2) ΔG\u003csub\u003egas\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u003csub\u003eele\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003evdw\u003c/sub\u003e (3) ΔG\u003csub\u003esol\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u003csub\u003eGB\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔG\u003csub\u003eSA\u003c/sub\u003e (4) (where ΔG\u003csub\u003ebind\u003c/sub\u003e is the binding free energy and G\u003csub\u003ecomplex\u003c/sub\u003e, G\u003csub\u003eprotein\u003c/sub\u003e and G\u003csub\u003eligand\u003c/sub\u003e are the free energies of complex, protein, and ligand, respectively. The sum of entropy contribution (TΔS) and enthalpy (ΔH) are for the binding free energy. The enthalpy (ΔH) includes the free energy of solvation (ΔG\u003csub\u003esol\u003c/sub\u003e) and free energy of the gas phase (ΔG\u003csub\u003egas\u003c/sub\u003e). ΔG\u003csub\u003esol\u003c/sub\u003e is composed of ΔG\u003csub\u003eSA,\u003c/sub\u003e the nonelectrostatic solvation component (nonpolar contribution) and ΔG\u003csub\u003eGB,\u003c/sub\u003e the electrostatic solvation energy (polar contribution). ΔG\u003csub\u003egas\u003c/sub\u003e includes ΔE\u003csub\u003eele\u003c/sub\u003e (electrostatic) and ΔE\u003csub\u003evdw\u003c/sub\u003e (van der Waals) energies, which exhibits the gas-phase interaction energy between protein and ligand.)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Acute toxicity\u003c/h2\u003e \u003cp\u003eThe acute toxicity of the lipo-diterpenoid alkaloids to animal subjects were investigated on the normal mice by using the aconitine linoleate (\u003cb\u003e1\u003c/b\u003e). Briefly, 110 kuming mice (aged 4-5 weeks, weighed 18-22 g, sex in half) were divided randomly into 11 groups, including one negative control group (dilute hydrochloric acid solution, pH 5.4), five dose groups (220, 210, 200, 190, 180 mg/Kg) of aconitine linoleate, and five dose groups (0.6, 0.55, 0.5, 0.4, 0.3 mg/Kg) for the positive control aconitine. All the groups were given the samples by intraperitoneal injection once time. The animals were observed for toxic signs for 4 h after the drug injection. And the changes in physical appearance, injury, pain and signs of illness were recorded daily for the 14 days. Finally, the mice were sacrificed after 14 days and the major tissues, including hart, spleen, kidney, liver, lung and thymus were excised for the analysis of macroscopic pathologicalanatomy. Then the tissues, fixed in formaldehyde 37%, were taken for microscopic evaluation using haematoxylin and eosin staining in the Pathology Laboratory of College of veterinary medicine, Sichuan Agricultural University. The pathological changes of the tissues were assessed and compared.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cem\u003e\u0026nbsp;3.1 Biology\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTaking adriamycin and etoposide as positive control, the \u003cem\u003ein vitro\u003c/em\u003e anti-proliferative activity\u0026nbsp;of Aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) was examined against MCF-7 and MCF-7/ADR cell lines by MTS method\u0026nbsp;(Ahmed et al., 2017). The results were expressed as IC\u003csub\u003e50\u003c/sub\u003e (median growth inhibitory concentration) values, representing the concentrations needed to produce a 50% inhibition of cell growth after incubation for 48 h (Table 1 and Figure\u0026nbsp;2)\u0026nbsp;(Luan et al., 2021).\u0026nbsp;In order to further explore the selectivity between\u0026nbsp;\u003cstrong\u003e1\u003c/strong\u003e and normal cells,\u0026nbsp;we used\u0026nbsp;NIH3T3\u0026nbsp;cell line\u0026nbsp;as normal cells for comparison and found that the IC\u003csub\u003e50\u003c/sub\u003e was\u0026nbsp;72.08\u0026plusmn;0.15\u0026nbsp;\u0026mu;M, and selective index (SI)=9.51 and 10.26\u0026nbsp;respectively compared with MCF-7 and MCF-7/ADR\u0026nbsp;(Table 1).\u003c/p\u003e\n\u003cp\u003eTo explore the effect of the Aconitine linoleate on cell proliferation of MCF-7/ADR cells, we analyzed cell cycle distribution of aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) at its IC\u003csub\u003e50\u003c/sub\u003e concentration\u0026nbsp;(Table 1)\u0026nbsp;using flow cytometry assay. Our data in Figure\u0026nbsp;3\u0026nbsp;shows that aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e), containing 8-linoleic acid side chain, 3-OH and N-ethyl group, exhibits the potent anti-proliferative effect on MCF-7/ADR cells by increasing percentage of cells G\u003csub\u003e0\u003c/sub\u003eG\u003csub\u003e1\u0026nbsp;\u003c/sub\u003ephase, decreasing the same in S phase.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 Molecular docking\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo better understand the potential binding targets of lipo-diterpenoid alkaloids, the molecular docking of aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) with 18 kinds of common antitumor targets was first screened by Discovery Studio 3.5.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe docking results in Table 2, indicated the most favorable target would be Topo\u0026nbsp;II\u0026nbsp;for the highest Libdockscore\u0026nbsp;(324.932). Interestingly, Topo\u0026nbsp;II\u0026alpha; may be the selective target (Libdockscore of 102.535), rather than Topo\u0026nbsp;II\u0026beta;, which\u0026nbsp;has been\u0026nbsp;failed in docking process.\u0026nbsp;In contrast, aconitine (\u003cstrong\u003e4\u003c/strong\u003e) could not successfully dock with those two targets (Table 3),\u0026nbsp;relating to the bad anti-proliferative activity of aconitine against those two cancer cells.\u003c/p\u003e\n\u003cp\u003eThe potential binding mode between compound \u003cstrong\u003e1\u003c/strong\u003e and Topo II\u0026alpha; has been further explored by molecular docking and molecular dynamic simulations, using the AutoDock vina 1.1.2 and Amber14 software. Bonding models of\u0026nbsp;\u003cstrong\u003e1\u003c/strong\u003e and Topo\u0026nbsp;II\u0026alpha; were depicted in Figure\u0026nbsp;4, showing the residue A/Arg-487 close to the phenyl group of the compound \u003cstrong\u003e1\u003c/strong\u003e by cation-\u0026pi; interaction (Figure\u0026nbsp;4A). To gain more information about the residues surrounding the binding site and their contribution to the whole system, the MMGBSA approach\u0026nbsp;was used\u0026nbsp;to calculate the contribution of the electrostatic, solvation, Van der Waals force, and residues to the free energy of binding.\u0026nbsp;Each residue interaction free energy was presented as electrostatic (∆\u003cem\u003eE\u003csub\u003eele\u003c/sub\u003e\u003c/em\u003e), solvation (∆\u003cem\u003eE\u003csub\u003esol\u003c/sub\u003e\u003c/em\u003e), \u003cem\u003eVan der Waals\u003c/em\u003e (∆\u003cem\u003eE\u003csub\u003evdw\u003c/sub\u003e\u003c/em\u003e) and total contribution (∆\u003cem\u003eE\u003csub\u003etotal\u003c/sub\u003e\u003c/em\u003e). In the Topo II\u0026alpha;-\u003cstrong\u003e1\u003c/strong\u003e complex, the residue A/Arg-487 has a strong electrostatic (∆\u003cem\u003eE\u003csub\u003eele\u003c/sub\u003e\u003c/em\u003e) contribution, with the ∆\u003cem\u003eE\u003csub\u003eele\u003c/sub\u003e\u003c/em\u003e of \u0026lt; -11.0 kcal/mol (Figure\u0026nbsp;4B). In addition, due to the close proximity between the nucleotides and the compound \u003cstrong\u003e1\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003estrong \u003cem\u003eVan der Waals\u003c/em\u003e interactions (\u0026lt; -4.5 kcal/mol) between nucleotides C/DC-8, D/DT-9, F/DA-12 and F/DG-13, and the ligand have been detected. Overall, the majority of the decomposed energy interaction originated from \u003cem\u003eVan der Waals\u003c/em\u003e interactions, mainly through hydrophobic interactions, such as A/Met-762, A/Met-766 and B/Ala-801.\u0026nbsp;It referred that the introduction of hydrophobic groups, such as halogen, nitro, benzene ring, etc., into the molecule, may increase the binding force with\u0026nbsp;Topo II\u0026alpha;, thereby enhancing its anti-proliferative activity.\u0026nbsp;Furthermore, the calculation of the total binding free energy for the Topo II\u0026alpha;-\u003cstrong\u003e1\u003c/strong\u003e complex led to an estimated ∆G\u003cem\u003e\u003csub\u003ebind\u003c/sub\u003e\u003c/em\u003e of -9.1 kcal/mol for \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003e(Table 4), indicating the strongly bind of \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003eto the binding site of the Topo II\u0026alpha;. This suggests that the anti-proliferative activity of\u0026nbsp;\u003cstrong\u003e1\u003c/strong\u003e is very likely to take effect through the complex with\u0026nbsp;Topo II\u0026alpha;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe docking results of 20-ns molecular dynamics simulation confirmed the preferential binding mechanism of Topo II\u0026alpha; and \u003cstrong\u003e1\u003c/strong\u003e. The root mean square deviation\u0026nbsp;(RMSD)\u0026nbsp;values of the protein skeleton basing on the initial structure with the simulation time was performed to figure out the dynamic stability of the models and ensure the rationality of the sampling strategy (Figure\u0026nbsp;4C), which proved the stability of protein in two systems under the simulation. The flexibility of the whole protein residues in Topo II\u0026alpha;-1 complex and free Topo II\u0026alpha; were detected by the root mean square fluctuations (RMSF) values (Figure 4D), presenting the difference in flexibility between the binding site of Topo II\u0026alpha; with \u003cstrong\u003e1\u003c/strong\u003e. The results presented a small degree of flexibility with a RMSF of less than 3 \u0026Aring; at the binding site, referring these residues seem to be more rigid as a result of binding to \u003cstrong\u003e1\u003c/strong\u003e. In summary, the rational explanation of the interactions between \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003eand Topo\u0026nbsp;II\u0026alpha; by\u0026nbsp;the molecular dynamic simulations provided valuable information for further development of the Topo\u0026nbsp;II\u0026alpha;\u0026nbsp;inhibitors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to prove the molecular docking results, the activity inhibition of topoisomerase enzyme with compound \u003cstrong\u003e1\u003c/strong\u003e was suspected as expecting mechanism for the action of lipo-diterpenoid alkaloids.The assay was performed to evaluate the Topo\u0026nbsp;I,\u0026nbsp;II\u0026alpha; inhibitory activity (Figure\u0026nbsp;5). Aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) exhibited promising inhibition activity below 200 \u0026mu;M, comparable to the known potent Topo\u0026nbsp;II\u0026alpha; inhibitor, etoposide. Whereas, it exhibited no obvious inhibition against Topo\u0026nbsp;I, even at the concentration of 200 \u0026mu;M. Thus, it was proved that compound \u003cstrong\u003e1\u003c/strong\u003e exerted its anti-proliferative activity through selective Topo\u0026nbsp;II\u0026alpha; inhibition, basing the results of both molecular docking and inhibition of topoisomerase activity.\u0026nbsp;However, in the Topo\u0026nbsp;II\u0026alpha; inhibition assay, the IC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues were much higher than in the cytotoxicity assays. It can be concluded that compound \u003cstrong\u003e1\u003c/strong\u003e possessed the anti-proliferative activity partially dependent on the Topo\u0026nbsp;II\u0026alpha; inhibition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 Acute toxicity\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor further application, the toxicity evaluation should be explored as an important factor. To investigate the toxicity of lipo-diterpenoid alkaloids, the acute toxicity test of aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) was carried out in Kuming mice for the LD\u003csub\u003e50\u003c/sub\u003e (half lethal dose) value. The results showed that the LD\u003csub\u003e50\u003c/sub\u003e value of aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) was\u0026nbsp;2.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e nmol/Kg. Compared with aconitine (LD\u003csub\u003e50\u003c/sub\u003e=4.5\u0026times;10\u003csup\u003e2\u003c/sup\u003e nmol/Kg), the safe dosage of aconitine linoleate to mice is\u0026nbsp;489\u0026nbsp;times higher than aconitine, suggesting the obvious toxicity reduction for lipo-diterpenoids (Table 5). Furthermore, as shown in Figure\u0026nbsp;6, after aconitine linoleate treatment, no obvious pathological lesions appear in the main tissues, including hart, spleen, kidney, liver, lung and thymus. The pathological morphology in mice tissue with/without compound \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003e(2.2\u0026times;10\u003csup\u003e5\u003c/sup\u003e nmol/Kg) or aconitine (4.5\u0026times;10\u003csup\u003e2\u003c/sup\u003e nmol/Kg) were also conducted by H\u0026amp;E staining method. Compared with the blank group, only liver and lung tissue in the aconitine linoleate group showed slight pathological changes, which are much milder than the aconitine group. For liver tissue, the obvious damage is found in the aconitine group with moderate diffuse vacuolar degeneration and punctate necrosis foci, which are occasionally seen in the aconitine linoleate group. For lung tissue, the alveolar septum is significantly thicker and the number of alveoli is decreased in the aconitine group; whereas, the alveolar septum in the aconitine linoleate group is slightly thicker, significantly better than that of the aconitine group.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eFirstly, compared with aconitine (4),\u0026nbsp;aconitine linoleate (1) exhibited high\u003cem\u003e\u0026nbsp;in\u003c/em\u003e \u003cem\u003evitro\u003c/em\u003e cytotoxic activities against both MCF-7 and MCF-7/ADR cell lines, and\u0026nbsp;displayed much lower toxicity in the in \u003cem\u003evivo\u003c/em\u003e acute toxicity evaluation in mice. Based on the results of molecular docking, aconitine linoleate (1) selectively well docked with Topo II\u0026alpha;, while\u0026nbsp;aconitine (4) failed. Considering the tumor targeting effect of long chain fatty acids\u0026nbsp;(Sauer et al., 2000), it is concluded that the introduction of 8-lipo group significantly enhanced its\u0026nbsp;affinity and selectivity for Topo II\u0026alpha;, as well as increasing the tumor targeting which led to the low toxicity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSecondly, according to the data of flow cytometry assay, aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) exhibits cytotoxic effect on MCF-7/ADR cells by increasing percentage of cells G0/G1 phase, decreasing the same in S phase. As everyone knows, Topo\u0026nbsp;II\u0026alpha;\u0026nbsp;and II\u0026beta; are two types of Topo II in humans. The expression level of Topo\u0026nbsp;II\u0026alpha;\u0026nbsp;is the highest in G2/M phase, followed\u0026nbsp;by S phase, and lowest in\u0026nbsp;G0/G1 phase, but that of Topo II\u0026beta; is always constant\u0026nbsp;(Jun et al., 2015). Therefore, due the selective inhibition of Topo\u0026nbsp;II\u0026alpha;,\u0026nbsp;aconitine linoleate (\u003cstrong\u003e1\u003c/strong\u003e) targeted the tumor cells in S phase.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThirdly, as a widely used chemotherapeutic agent, doxorubicin, mainly targeting Topo II\u0026alpha; (Du et al., 2011), has caused drug-resistance during clinical application, which is a complex process (Ganapath and Ganapathi, 2013). Different resistant mechanisms have been recognized, including the enhanced expression of the P-glycoprotein, ABCB1, ABCG2, the down-expression level of topo II\u0026alpha;, overexpression of multidrug resistance protein 1(MDR1), and even the activation of autophagy (Mechetner et al., 1998, Latorre et al., 2012, Shatha AbuHammad and Zihlif, 2013, Guo et al., 2016). Although targeted to the same enzyme, aconitine linoleate (1) is mainly a topo catalytic inhibitor, which could selectively inhibit Topo II\u0026alpha;, rather than a topo poison like doxorubicin. They may have different topo-inhibit mechanism. Moreover, the reversal mechanism of tumor drug resistance of aconitine linoleate (1) should be a multi-targeted way, which need further explore in the next step.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe conclude that aconitine linoleate (\u003cb\u003e1\u003c/b\u003e) not only selectively inhibits the expression and activity of Topo IIα, but also induce a significant increase in the percentage of cells at G\u003csub\u003e0\u003c/sub\u003eG\u003csub\u003e1\u003c/sub\u003e phase, leading its excellent anti-proliferative activity against MCF-7 and MCF-7/ADR \u003cem\u003ein vitro\u003c/em\u003e. Since the introduction of 8-lipo group, \u003cb\u003e1\u003c/b\u003e is much less toxic than aconitine. Lipo-alkaloid would be a powerful and potentially anti-tumor compound against drug-resistant breast cancer, bringing more possibilities for fighting breast cancer in the future.\u003c/p\u003e"},{"header":"Associated Content","content":"\u003cp\u003eSupporting Information\u003c/p\u003e\n\u003cp\u003eThe Supporting Information is available free of charge on website\u0026nbsp;at DOI:\u003c/p\u003e\n\u003cp\u003eData availability \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs supplemental material and upon request.\u003c/p\u003e\n\u003cp\u003eAccession Codes\u003c/p\u003e\n\u003cp\u003eThe PDB access code for structure of 1 bond to topoisomerase II\u0026alpha; is 5GWK. Authors will release the atomic coordinates and experimental data upon article publication.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCompeting interests\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\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe animal use protocol listed below has been reviewed and approved by the Sichuan Agricultural University Animal Ethical and Welfare Committee.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors approved the manuscript to be published.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eShangxian Luan: Writing paper and participating in Molecular Docking and \u003cem\u003ein vitro\u003c/em\u003e anti-proliferative activity, acute toxicity. \u0026nbsp;Yingying Gao: Participating in \u003cem\u003ein vitro\u003c/em\u003e cell cycle analysis and Topo inhibition assay. Xiaoxia Liang: Designing and leading experiments, writing and revising the paper. Li Zhang: \u0026nbsp;participating in acute toxicity. Qiang Wu: participating in acute toxicity. Yunkai Hu: participating in acute toxicity. Shixi Liu: participating in Molecular Docking. Lizi Yin\u0026nbsp;and\u0026nbsp;Changliang He guided the experiment.\u0026nbsp;The authors declare that all data were generated in-house and that no paper mill was used.\u003c/p\u003e\n\u003cp\u003eFunding \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China Youth Fund and the National Natural Science Foundation of China (Grant No. 81703387, 31972743)\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAbdelhaleem EF, Abdelhameid MK, Kassab AE, Kandeel MM (2018) Design and synthesis of thienopyrimidine urea derivatives with potential cytotoxic and pro-apoptotic activity against breast cancer cell line MCF-7. 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Journal of Natural Medicines 74: 83-89.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11418-019-01346-z\"\u003ehttps://doi.org/10.1007/s11418-019-01346-z\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eYamashita H, Takeda K, Haraguchi M, Abe Y, Kuwahara N, Suzuki S, Terui A, Masaka T, Munakata N, Uchida M, Nunokawa M, Kaneda K, Goto M, Lee K-H, Wada K (2018) Four new diterpenoid alkaloids from Aconitum japonicum subsp. subcuneatum. Journal of Natural Medicines 72: 230-237.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11418-017-1139-9\"\u003ehttps://doi.org/10.1007/s11418-017-1139-9\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003eYamashita H. \u0026nbsp;MM, Hiramori K., \u0026nbsp;Kobayashi D., Suzuki Y., Mizukami M. , Goto M. ,Lee K.‑H. , Wada K. (2020) Cytotoxic diterpenoid alkaloid from Aconitum japonicum subsp. subcuneatum.\u0026nbsp;Journal of Natural Medicines 74: 83-89.\u0026nbsp;\u003ca href=\"https://doi.org/10.1007/s11418-019-01346-z\"\u003ehttps://doi.org/10.1007/s11418-019-01346-z\u003c/a\u003e\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 1 \u003cspan style=\"background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eThe IC\u003csub\u003e50\u003c/sub\u003e (\u0026mu;M) results of compounds 1 and 4 against MCF-7,\u003c/span\u003e\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026nbsp;MCF-7/ADR\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size: 13px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026nbsp;\u003c/span\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eand\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026nbsp;NIH3T3\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width:100.0%;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNo.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eMCF-7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eMCF-7/ADR\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 13px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eNIH3T3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;border:none;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;border:none;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e7.58\u0026plusmn;0.06\u003csup\u003ea,b\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e7.02\u0026plusmn;0.12\u003csup\u003e\u0026nbsp;a,b\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e72.08\u0026plusmn;0.15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026gt;400\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e86.35\u0026plusmn;0.32\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;a,b,\u003c/span\u003e\u003c/sup\u003e\u003csup\u003e\u003cspan style=\"font-size:15px;\"\u003ec\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eAdriamycin\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1.22\u0026plusmn;0.43\u003csup\u003e\u0026nbsp;a,b,c\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e67.61\u0026plusmn;6.50\u003csup\u003e\u0026nbsp;b,\u003c/sup\u003e\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size:15px;\"\u003ec\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEtoposide\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e18.01\u0026plusmn;1.64\u003csup\u003e\u0026nbsp;a,b,c\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e35.48\u0026plusmn;0.29\u003csup\u003e\u0026nbsp;a,\u003c/sup\u003e\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size:15px;\"\u003ec\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:23.22%;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eSI\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:27.16%;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:8.5pt;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e9.51\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.16%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.44%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e10.26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-top:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eNote: Compared with positive control group (\u003csup\u003ea\u003c/sup\u003eAdriamycin,\u0026nbsp;\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026nbsp;b\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eEtoposide)\u003csup\u003e\u0026nbsp; a\u003c/sup\u003eP\u003c/span\u003e\u003cspan style=\"font-size: 15px; font-family: Symbol; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026lt;\u003c/span\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e0.05,\u003csup\u003e\u0026nbsp;b\u003c/sup\u003eP\u003c/span\u003e\u003cspan style=\"font-size: 15px; font-family: Symbol; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026lt;\u003c/span\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e0.05;\u0026nbsp;Compared with \u003cstrong\u003e1\u003c/strong\u003e group, \u003csup\u003ec\u003c/sup\u003eP\u003c/span\u003e\u003cspan style=\"font-size: 15px; font-family: Symbol; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026lt;\u003c/span\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e0.05; - means no data,\u003c/span\u003e\u003csup\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eSI:\u0026nbsp;selectivity of compound 1 between\u003c/span\u003e\u003cspan style='font-size: 13px; font-family: \"Arno Pro\", serif; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;'\u003e\u0026nbsp;MCF-7\u0026nbsp;\u003c/span\u003e\u003cspan style=\"font-size: 15px; background-position: 0% 0%; background-repeat: repeat; background-attachment: scroll; background-image: none; background-size: auto; background-origin: padding-box; background-clip: border-box;\"\u003eor MCF-7/ADR and NIH3T3 cells\u003c/span\u003e\u003cspan style=\"font-size:15px;\"\u003e.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-top:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 2 Docking results of aconitine linoleate\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;(\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e1) and receptor protein molecules\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 100%;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eReceptor protein\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePDB Number\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eLibdockscore\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePoses\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅱ\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e5bTc\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e324.932\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅰ\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1nh3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e83.3349\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEpidermal Growth Factor HER2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e3WSQ\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e147\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEpidermal Growth Factor HER3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e5O4O\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e142\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eMatrix metalloproteinases\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1bqo\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e109.144\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ehuman\u0026nbsp;basic\u0026nbsp;fibroblast\u0026nbsp;growth\u0026nbsp;factor\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1bfb\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eHuman vascular endothelial growth factor\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1vpf\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eProtein kinase C\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e2kf9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eInsulin receptor\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e3ekk\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePlatelet-derived growth factor receptor\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e3mjg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e144.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e56\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eCytochrome P450 3A4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4i3q\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e133.45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eAngiogenin 1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4k0v\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e138.278\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eAngiogenin 2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4iml\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e132.827\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e32\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eHuman tyrosine kinase c-KIT\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e6gqj\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e121.48\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eHuman cyclooxygenase Ⅱ\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e5f19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 48.34%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEpidermal growth factor receptor\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e1ivo\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20.04%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e120\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.62%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNote: LibDockscore indicates the degree of docking between receptors and ligands; Poses represents the way ligands bind to proteins; If the score and poses are 0, the docking failed.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 3 Comparation of the docking results of between aconitine linoleate\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e(\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e)\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eand aconitine (4) with receptor protein Topo Ⅱ\u0026alpha; and Topo Ⅱ\u0026beta;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"width: 100%;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21.68%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eCompound\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.34%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eReceptor protein\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePDB Number\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eLibdockscore\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.98%;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePoses\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.68%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.34%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅱ\u0026alpha;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4fm9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e102.535\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.98%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:11.0pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.34%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅱ\u0026beta;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4g0w\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.98%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 21.68%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.34%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅱ\u0026alpha;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4fm9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.98%;border: medium none;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.34%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTopo Ⅱ\u0026beta;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4g0w\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 20%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.98%;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 8.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNote: LibDockscore indicates the degree of docking between receptors and ligands; Poses represents the way ligands bind to proteins; If the score and poses are 0, the docking failed.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 4. Average binding free energies (kcal mol\u003csup\u003e\u0026minus;1\u003c/sup\u003e) for the Topo2\u0026alpha;-1 complex along with the different energy contributions\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;'\u003e\n \u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eCompd\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;Evdw\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;Eele\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;Ggas\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;EGB\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;GSA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;Gsol\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;H\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eT\u0026Delta;S\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1pt medium;padding: 0in 5.4pt;height: 11.1pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026Delta;Gbind\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 41.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eL29\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-99.8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-45.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-145.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 37.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e88.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-11.0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e77.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-67.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-58.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 41.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.95pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e-9.1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.5pt;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 5.\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003eAcute toxicity test results of compound\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;1 and 4 in mi\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;\"\u003ece through celiac injection\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.15pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eCompound\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eConcentration (mg/Kg)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.2pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNumber of deaths\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103.45pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003eLD\u003csub\u003e50\u003c/sub\u003e (nmol/Kg)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.15pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 4.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size:15px;color:red;\"\u003e1\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 4.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e180\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.2pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 4.9pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103.45pt;border-color: windowtext currentcolor currentcolor;border-style: solid none none;border-width: 1pt medium medium;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 4.9pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.15pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e190\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.2pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103.45pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.15pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n 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6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e0.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 99.2pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;\"\u003e9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103.45pt;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 92.15pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 99.2pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 103.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 6.25pt;vertical-align: top;\"\u003e\n \u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;text-align:justify;'\u003e\u003cspan style=\"font-size:15px;color:red;\"\u003e4.5\u0026times;10\u003csup\u003e2\u003c/sup\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;font-size:16px;font-family:\"Times New Roman\",serif;margin-bottom:6.0pt;text-align:justify;text-indent:10.45pt;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNote: A total of 11 groups including the negative control group (dilute hydrochloric acid solution, pH 5.4), each with 10 mice; There was no death in the negative control group.\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Aconitine linoleate, cytotoxic activity, topoisomerase IIα inhibitor, acute toxicity, MCF-7 and MCF-7/ADR breast cancer cells, Molecular modeling","lastPublishedDoi":"10.21203/rs.3.rs-1005235/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1005235/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAconitine linoleate (\u003cb\u003e1\u003c/b\u003e) is a lipo-diterpenoid alkaloid, isolated from \u003cem\u003eAconitum sinchiangense\u003c/em\u003e W. T. Wang. The study aimed at investigating the anti-proliferative efficacy and the underlying mechanisms of \u003cb\u003e1\u003c/b\u003e against MCF-7 and MCF-7/ADR cells, as well as obvious the safety evaluation \u003cem\u003ein vivo\u003c/em\u003e. The cytotoxic activities of \u003cb\u003e1\u003c/b\u003e were measured \u003cem\u003ein vitro.\u003c/em\u003e Also, we investigated the latent mechanism of \u003cb\u003e1\u003c/b\u003e by cell cycle analysis in MCF-7/ADR cells, and Topo I, Topo IIα inhibition assay. Molecular docking is done by Discovery Studio 3.5 and Autodock vina 1.1.2. Finally, the acute toxicity of \u003cb\u003e1\u003c/b\u003e was detected on mice. \u003cb\u003e1\u003c/b\u003e exhibited significant anti-tumor activity against both MCF-7 and MCF-7/ADR cells, with IC\u003csub\u003e50\u003c/sub\u003e value of 7.58 and 7.02 \u0026micro;M, which is 2.38 times and 5.05 times more active, respectively than etoposide in both cell lines, and being 9.63 times more active than adriamycin in MCF-7/ADR cell lines. The molecular docking and topo inhibition test found that it's a selective inhibitor of topoisomerase Ⅱα. Moreover, activation of damage response pathway of the DNA leads to cell cycle arrest at G\u003csub\u003e0\u003c/sub\u003eG\u003csub\u003e1\u003c/sub\u003ephase. Furthermore, the \u003cem\u003ein vivo\u003c/em\u003e acute toxicity of \u003cb\u003e1\u003c/b\u003e in mice displayed lower toxicity than aconitine, with LD\u003csub\u003e50\u003c/sub\u003e of 2.2\u0026times;10\u003csup\u003e5\u003c/sup\u003enmol/Kg and only slight pathological changes in liver and lung tissue, 489 times safer than aconitine. In conclusion, compared with aconitine, \u003cb\u003e1\u003c/b\u003e has more significant anti-proliferative activity against MCF-7 and MCF-7/ADR cells, and greatly reduces \u003cem\u003ein vivo\u003c/em\u003e toxicity, which suggesting this kind of lipo-alkaloids are powerful and promising antitumor compounds for breast cancer.\u003c/p\u003e","manuscriptTitle":"Aconitine linoleate, a natural lipo-diterpenoid alkaloid, stimulates anti-proliferative activity reversing doxorubicin-resistant in MCF-7/ADR breast cancer cells as a selective topoisomerase IIα inhibitor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-10-22 13:50:29","doi":"10.21203/rs.3.rs-1005235/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2021-10-09T12:16:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"961e6c99-8da4-462f-b619-0a839fefb647","owner":[],"postedDate":"October 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":8026916,"name":"Clinical Pharmacology"}],"tags":[],"updatedAt":"2021-10-22T13:50:29+00:00","versionOfRecord":[],"versionCreatedAt":"2021-10-22 13:50:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1005235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1005235","identity":"rs-1005235","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}