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  Carnosol (Figure 1) is a naturally occurring phytopolyphenol found in Rosemary officinalis. Its herbs and oils are commonly used as a spice and flavoring agents in food processing due to its desirable flavor and high antioxidant activity^[1]. Carnosol has been evaluated for anti-cancer property in prostate, breast, skin, leukemia, and colon cancer with promising results. These studies have provided evidence that carnosol targets multiple deregulated pathways associated with inflammation and cancer that include nuclear factor kappa B (NF-κB), apoptotic related proteins, phosphatidylinositol-3-kinase (PI3 K)/Akt, androgen and estrogen receptors, as well as molecular targets. In addition, carnosol appears to be well tolerated in that it has a selective toxicity towards cancer cells versus non-tumorigenic cells and is well tolerated when administered to animals^[2].

Figure 1: Structures of Carnosol and Resvaratrol

  Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (Figure1) is a stilbenoid, a type of natural phenol, and a phytoalexin produced naturally by several plants when under attack by pathogens such as bacteria or fungi. Resveratrol is also found in the skin of red grapes and in other fruits. Resveratrol has also been produced by chemical synthesis^[3] or by biotechnological synthesis (metabolic engineered micro-organisms)^[4] and is sold as a nutritional supplement derived primarily from Japanese knotweed. In mouse and rat experiments, anti-cancer, anti-inflammatory, blood-sugar-lowering and other beneficial cardiovascular effects of resveratrol have been reported. These results have yet to be replicated in humans.

  There is a profound need to explore and identify the anti-cancer properties of these two (carnosol, resvaretrol) medicinal herbs. These two compounds (carnosol, resvaretrol) have under various phases of research in individually. But the results are not satisfactory in their unique actions with high doses. Now our experiment could expect to limit the high dose criteria of these photochemical and more efficacies by combined action in rat models for cancer comprehensiveness.

  Breast cancer is the third most common tumor in the world and represents 9% of the global cancer burden. This percentage varies considerably around the world: in high-risk areas, such as North America and western Europe, breast cancer accounts for one in four female cancers, while in low-risk areas such as China and Japan, it accounts for only one in eight to one in 16. The importance of environmental factors in the etiology of breast cancer is demonstrated by the change in risk in migrant populations. Risks factors include family history, high fat diet, early age of menarche, nulliparity and late age of menopause. In India one study saying that, in the city of Bombay there is an alarming rise of cases of breast cancer in women. It will double by 2025. An average of 1,300 cases was recorded in Mumbai every year between 2001 and 2005. According to a study by Tata Memorial Hospital, where population-based Mumbai Cancer Registry data was studied in women between the age group of 25 and 74, it was observed that cases of breast cancer have been increasing among older women (above 50 years) than the younger age group^[5].

  NF-κB is able to induce several of cellular alterations and has been shown to be constitutively activated in many types of cancer cells. There are several mechanisms by which NF-κB transcription factors are uncoupled from their normal modes of regulation, and these have been associated with cancer. Constitutively activated NF-κB transcription factors have been associated with several aspects of tumorigenesis, including promoting cancer-cell proliferation, preventing apoptosis and increasing a tumors angiogenic and metastatic potential. NF-κB site is present within the cyclin D1 promoter^[6] and there is strong evidence that NF-κB dependent cyclin D1 is over-expressed in breast cancers. Leukemia and lymphoma-cancers of the bone marrow and lymph nodes respectively are caused by uncontrolled proliferation of blood cells. Numerous studies have documented elevated or constitutive NF-κB DNA binding activity both in mammary carcinoma cell lines and primary breast cancer cells of human and rodent origin^[7,8]. Here we focused on the interactions of NF-κB with carnosol and resveratrol both independently and combinational by molecular docking interaction studies. This can be achieved by using various computational tools and techniques. The independent binding energies of carnosol and resveretrol with NF-κB showed effective inhibition energies further it may inhibit the activation of NF-κB as per the literature it is also evidenced. As of thought use of combination of these two may enhance the binding energies to NF-κB due to different binding sites and also decreases the criteria of high dose in use of phytochemicals. This clearly urges the scope for combinational natural drug research for cancer comprehensiveness.

Methods

  In present work all the calculations were carried out with high frequency computational analysis such as molecular modeling, energy minimizations, design and optimization of lead molecules, protein ligand interaction studies by molecular docking , dynamics etc., in Hi-end server (Pentium IV 3.4 MHzs, AMD Athlon 64 bit, Dual processor with1GB RAM) manufactured by HCL Corporation, Pondicherry, India was used. Most of the software used was either Windows or Linux flat form based which were well accepted and referred in various publications at high rated research journals. Academic license was obtained for the commercial software used in the present study by requesting the concerned suppliers. The software used in the present study a PyMOL(https://www.delanoscientific.com/), Autodock-Tool (https://autodock.scripps.edu/)^[9], National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/), Protein Data Bank (PDB) (www.rcsb.org/pdb), PROCHECK, ProSA-web^[10] (https://prosa.services.came.sbg.ac.at), What-if , PDBSUM^[11] (https://www.ebi.ac.uk/pdbsum/), ADME tox Server: (https://ilab.acdlabs.com/iLab2/index.php#), PRODRG (www.davapc1.bioch.Dungee.ac.uk), HEX Docking Server.

Preparation of files for docking
   PDB file for docking are prepared by editing retrieved mouse NF-κB structure from RCSB(PDB-1VKF). Resvaratrol and Carnosol were designed by using PRODRG server^[12] and partial charges were added and saved as pdb files. The compounds were then tested for Lipinski’s Rule of Five using the Molinspiration server (https://www.molinspiration.com/). ADME toxicity of carnosol and resveretrol were predicted in ADMEtox server.

Docking of p50, p65 monomers with carnosol and resveretrol independently: AutoDock 4.0 (ADT) was used for the docking interactions of carnosol and resveretrol molecules on to the mouse p50, p65, monomers^[13]. In order to run ADT, to the PDB files of Mouse p50, p65, monomers, the histidine hydrogens as well as polar hydrogens were added and the C- and N-terminal ends were charged and the Kollman united atom partial charges were assigned. Furthermore the atomic salvation parameters were assigned and were saved in pdbqt format. Carnosol and resveretrol pdbqt file was obtained from PRODRG Server. All the atom types were checked in the carnosol and resveretrol and modified when needed. Carnosol and resveretrol were chosen charged and hydrogens were added in order to fill all empty valences, and the Gasteiger charges were calculated for the atoms, and were then saved. In order to run AutoDock, grid maps have to be calculated. This was done by using the module AutoGrid, for carnosol and with the same parameters: number of grid points in X, Y & Z were taken as default as 40Χ40Χ40 (this covers the active site extensively and let the carnosol move without any constraints regarding the box size), spacing between grid points, 0.375 Å and a common grid centre. The grid centre was chosen slightly of the centre axis of the active site in order to avoid any symmetry problems that might arise. In docking matrix the docking parameter files (dpf) were generated by a Python script that uses the methods in AutoDock. The script takes one pdbqt file, loops over the pdbqt files and sets the name of the maps and the carnosol in the parameter file. It also sets the Lamarckian genetic algorithm (LGA) to be used with a population size of 150 individuals. Thus 150 individuals were calculated at 100 different runs (i.e. 100 dockings) and the runs had two stop criteria: a maximum of 2,500,000 energy evaluations or a maximum of 27,000 generations. The carnosol and resveretrol were set to start in a random position and conformation, the translations were set to have a maximum of 2 Å/step and the quarterion and the tortion both had a maximum at 50°/step. The elitism number was set to 1. The mutation rate and the crossover rate were 0.02 and 0.80 respectively. The probability that an individual in the population will undergo a local search was set to 0.06 and the constraint used in the pseudo- solis and wets local search was set to a maximum of 150 iterations per search. The maximum number of successes or failures before changing rho in the local search method was both set to 4. The size of the local search space was set 1.0 and the smallest step the local search could take before ending was set to 0.01. These standardized docking parameters create a file for each ligand and hence AutoDock program for each ligand was run. Using the hardware as mentioned in earlier section, one run (a Docking) took between 7 and 30 minutes depending on the complexity of the Carnosol, number of rotatable and number of atoms. Docking results in graphical presentation were analyzed by using PMV (Python Molecular Viewer) 1.4.5.

Docking of NF-κB with carnosol and resvaratrol: Here we used same parameter set like mentioned above but instead of monomers here we used heterodimer (p50-p65). In Autodock4.2 suit docking has been done and DLG files are analyzed.

Complex docking with DNA
  For complex docking we took the docked complex dock log files (DLG) generated from Autodock of p50-p65 heterodimer with carnosol and resveretrol further subjected in HEX server for complex docking with DNA.

DNA docking with NF-κB-RESL-CAR complex: Complex docking analysis predicts the binding of resveretrol and carnosol at DNA binding pocket of NF-κB. The resveretrol- carnosol complex suggested to bind in the area occupied by the major groove at the site of a DNA binding regions, this binding mode of resveretrol carnosol complex would signiï¬