A Pre-clinical Trial Study: Anti-human Colon Cancer Effect of Thalassiolin B in vitro with Enzymes Inhibition Effects and Molecular Docking Studies.

In this study, it is recorded the inhibition effect of Thalassiolin B on aldose reductase, alpha-glucosidase and alpha-amylase enzymes. In the next step, the molecular docking method was used to compare the biological activities of the Thalassiolin B molecule against enzymes formed from the assembly of proteins. In these calculations, the enzymes used are Aldose reductase, Alpha-Amylase, and Alpha-Glucosidase, respectively. After the docking method, ADME/T analysis of Thalassiolin B molecule was performed to be used as a drug in the pharmaceutical industry. In the MTT assay, the anti-human colon cancer properties of Thalassiolin B against EB, LS1034, and SW480 cell lines were investigated. The cell viability of Thalassiolin B was very low against human colon cancer cell lines without any cytotoxicity on the human normal (HUVEC) cell line. The IC50 of the Thalassiolin B against EB, LS1034, and SW480 were 483, 252, and 236 µg/mL, respectively. Thereby, the best cytotoxicity results and anti-human colon cancer potentials of our Thalassiolin B were observed in the case of the SW480 cell line. Maybe the anti-human colon cancer properties of Thalassiolin B are related to their antioxidant effects.

It has been seen in many studies that the numerical values obtained from experimental and theoretical studies are quite compatible with each other 5,6 . Therefore, it has been observed that the theoretical calculations made before experimental studies will be more useful. As a result, it is possible to synthesize more effective and active molecules with theoretical calculations made before experimental studies 7 . The results of these calculations direct the experimental studies to be conducted. As a result, huge savings will be made for experimental studies. In theoretical calculations, the most widely used method is the molecular docking method. Many parameters are calculated due to calculations made with the molecular docking method. These parameters provide important information about the biological activities of molecules. After the docking calculations, ADME/T Absorption, Distribution, Metabolism, Excretion and Toxicity analysis of the molecule was performed. With the ADME/T analysis, the effects and reactions of drug molecules in human metabolism in cells and tissues are tried to be predicted theoretically 8 . These effects and responses are tried to be predicted by numerical values of the parameters obtained due to the calculations. Each parameter obtained is tried to be predicted the effect and response in different organs or tissues. These results give the properties of the molecule to be used as a drug in the future.
Cancer is a genetic disease that is ultimately the result of the effects of environmental factors. In 2010, more than 14,000,000 people were diagnosed with cancer and about 7,000,000, or 50 , died. Since last year, cancer has been ranked number one in the world in terms of mortality. Until now, cardiovascular disease has been the number one concern. The highest percentages of cancers are breast cancer, hepatocarcinoma, colorectal cancer, gastric cancer, and lung adenocarcinoma in women and ovarian cancer in men, respectively 9, 10 . The highest rates of cancer in children include blood, brain, and lymph nodes. The highest risk factor for cancer is aging. The older you get, the greater your risk of developing cancer. 93 of cancers are caused by the environment, 30 by cigarette smoke, 35 by diet, 25 by infectious diseases, and 10 by ion and non-ion rays 11 13 . Cancers are caused by a series of mutations in human genes and each mutation causes some new changes in the cell. Chemicals cause cancer cells called carcinogens. There are more than 100,000 types of chemicals in nature that directly or indirectly affect the cytoplasm and the nucleus of cells and lead to genetic disorders that cause mutant cup heads 13,14 . Various viruses, bacteria, and radiation, in turn, produce inherited cancers, which account for about 7 of all cancerous tissue: Blood, lymph nodes, sarcoma, carcinoma, embryonic cells, and germ cells. Cancer is a disease that disrupts intercellular relationships and disrupts vital and key genes 15 18 . These molecular irregularities affect the cell division cycle and lead to a lack of cell differentiation. Cancer can be treated in several ways: surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, or a combination of these. Due to the relative inefficiency and very severe side effects of chemotherapy drugs, researchers and scientists have been looking for new formulations of various compounds, especially antioxidant molecules 12,18 .
In the present study, we investigated the anti-colon cancer potentials of the Thalassiolin B against EB, LS1034, and SW480 cell lines. The antioxidant properties of the Thalassiolin B were calculated against DPPH free radicals. In this paper, we have evaluated that the in vitro inhibition effects of the Thalassiolin B on AR, alpha-glucosidase and alpha-amylase enzymes, also, molecular docking of enzymes results were studied.

Enzymes assays
Determination of the α-amylase inhibitory property of the ligands was carried out following procedures described by Adefegha et al. 19 and according to the previous studies 20 . Aldose reductase activity was measured according to the previous studies 21 and measured the decrease of NADPH at 340 nm spectrophotometrically using the DLglyceraldehyde substrate. Alpha-glycosidase inhibitory effect of Justiciresinol was evaluated using p-NPG as the substrate, according to the procedure of Tao et al. 22 and according to other study 23 .

Docking studies
Many studies show that the most commonly used method to compare the biological activities of molecules is molecular docking 24 . By molecular docking method, comparison of biological activities is made from the numerical value obtained from the interactions of molecules and enzymes 25 . The active areas of the molecules are determined by calculations. In this study, molecular docking calculations were made to compare the biological activities of the Thalassiolin B molecule. Molecular docking calculations to calculate the biological activity of the Thalassiolin B molecule were performed using the Maestro Molecular modeling platform version 12.2 by Schrödinger. Proteins and Thalassiolin B molecules must be prepared for these calculations. In docking calculations, a different process is performed for molecules at each stage. First, it was used from Gaussian software program 26 to obtain optimized structures of molecules, files with extension *.sdf were created using these structures. Using these files, all calculations were made with the Maestro Molecular modeling platform version 12.2 by Schrödinger, LLC 27 . The Maestro Molecular modeling platform version 12.2 by Schrödinger comes together from many modules. In the first module used, the protein preparation module 28 was used to prepare the studied proteins for calculations. It should be well known that the enzymes studied are composed of many small proteins. The crystal structures of these enzymes studied have been downloaded from the protein data bank site. The enzymes studied were first minimized and the water molecules in their crystal structures were removed. In the next step, the active sites of the enzymes were de-termined for calculations, in which freedom of movement was given to all proteins in this active site. Thus, these proteins have been enabled to interact more easily with molecules. In the next step, the LigPrep module 29 was used to prepare the working molecules for calculations.
Calculations were made to find 3D structures at physiological pH values of the Thalassiolin B molecule and high energy isomers in the correct protonation conditions. In the next step, the prepared protein and molecules were docked with each other. The Glide ligand docking module 30 was used for this step. In this module, the OPLS3e method was used in all calculations for docking calculations of molecules and proteins. Numerical values of many parameters obtained due to molecular docking calculations using this module are used. After the docking calculations, ADME/T analysis absorption, distribution, metabolism, excretion and toxicity was performed to examine the future drug properties of the molecule. The Qik-prop module 31 of the Schrödinger software was used for ADME/T analysis.

Assessment of antiproliferative effect by MTT assay
In this assay, following human colon cancer and normal cell lines were used to study the cytotoxicity and anticancer potential of human colon over the Thalassiolin B and Cisplatin As a common positive control using the common cytotoxicity test i.e., MTT assay in vitro condition: 1 Human colon cancer cell lines: EB, LS1034, and SW480. 2 Normal cell line: HUVEC. These cells were maintained in a DMEM medium with 10 bovine embryos and 1 penicillin/streptomycin antibiotic to prevent fungal growth . Prerequisites for cell growth at 37 are 5 CO 2 with 95 moisture, which was provided by the NÜVE incubator EC160 model . For MTT assay, when the cells reached at least 70 cell growth, they were separated from the bottom of the flask by trypsin-ethyldiamine tetraacetic acid and centrifuged at 1700 rpm for 6-1 minutes. Cell precipitate was prepared in suspension in 1 mL of culture medium. The viability of cells in cell suspension was determined by mixing it with an equal proportion of trypan blue and counting them with a neobar slide under a light microscope. After confirming that the cells were not infected, cells with a viability of more than 90 were used for testing 32 .
To investigate the effect of 2 -Hydroxy-5 -methoxyacetophenone on cancer cell proliferation, tetrazolium MTT salt colorimetric method was used. For this test, 10 4 cells were added to each 96-well plate well. After 24 hours of incubation, concentrations of 1-1000 μg/mL were treated on cancer and normal cells for 24, 48, and 72 hours. After these times, 20 μL of MTT solution and 200 μL of base culture medium were added to each well. The plate was placed in a dark CO 2 incubator at 37 for 4 hours in the dark. After this time, 100 microliters of DMSO were added to each well. 492 and 630 nm optical readings were placed in the ELISA reader DANA model DA3200 . The cell viability was computed by the following formula 32 :

Cell viability
Sample A Control A 100 2.4 Evaluation of the antioxidant property of the Thalassiolin B The ability of hydrogen atoms or electrons to give off different compounds and antioxidant molecules in this test is measured by the degree of decolorization of the 2 and 2-diphenyl-1-picryl-hydrazyl purple solution in methanol. In this method, DPPH Sigma-Aldrich was used as a stable radical compound. Thus, 100 μL of various dilutions of Thalassiolin B in methanol was added to 10 mL of 0.005 DPPH solution in methanol. After 1 hour of incubation at the absorption room temperature, the samples were read against Blank at 518 nm. The DPPH inhibition percentage was computed by the following formula 33 : In this formula, Control A shows the negative control of light absorption that lacks nanoparticles, and Sample A expresses the amount of light absorption of different concentrations of nanoparticles 33 .

Qualitative measurement
To compare the results, in addition to the formula mentioned above, which was calculated as an average of 5 repetitions of experiments. The results were analyzed using SPSS software version 22 and the statistical differences between the treatments were examined by t-test and p ≤ 0.01was considered significant.

Enzymes results
The important purpose of this study is to determine efficacious, selective and more potent inhibitors for aldose reductase, alpha-glucosidase and alpha-amylase enzymes. Hence, inhibition analysis of these enzymes was recorded with Thalassiolin B to control diabetic complications. Researchers often list an IC 50 value to describe inhibitory effects, but the Ki constant is the more suitable for measurement. Both the Ki and IC 50 parameters of compounds were performed in the current study from Lineweaver-Burk graphs 1/V-1/ S and Activity -Inhibitor graphs respectively. IC 50 and Ki values were calculated for AR, alpha-glucosidase and alpha-amylase. The results for inhibitory activity of the studied Thalassiolin B compound are shown in Table 1

Molecular modeling results
The biological activities of molecules are compared with many parameters obtained by the Maestro program, which is used to calculate the biological activities of molecules 34 . With this method, many parameters are calculated due to the interaction of molecules with enzymes 35 . By using these calculated parameters, the biological activities of molecules with other molecules are compared. Enzymes used for this comparison are Aldose reductase PDB ID: 3V36 AR , Alpha-Amylase PDB ID: 3BAJ Alpha-Amy , and Alpha-Glucosidase PDB ID: 3TOP alpha-Gly , respectively. The parameters obtained due to interactions with these enzymes are given in Table 2.
The most important parameter among the many parameters obtained for the Thalassiolin B molecule due to molecular docking calculations is the Docking score parameter of the Thalassiolin B molecule 36 . This parameter obtained is the numerical value of the interactions between Thalassiolin B molecule and enzyme. Due to calculations, the molecule with the most negative numerical value of this parameter has higher biological activity than other molecules. It should be well known that the more interaction between any molecule and enzyme, the more the numerical value of this parameter decreases. Therefore, the most important factor affecting the biological activities of molecules is the interactions between molecules and proteins in enzymes. These interactions have many interactions such as hydrogen bonds, polar and hydrophobic interactions, π-π and halogen 37 . These interactions are given in Figs. 1,  2, and 3.
Many parameters obtained from the docking calculations of the Thalassiolin B molecule were obtained. Each parameter evaluates the interaction between molecule and enzyme from different angles. The Glide hbond, Glide   39 . Each parameter evaluates the interaction between molecule and enzyme from different perspectives. Another important parameter is the QPlogHERG, which is the numerical value of the estimated IC 50 value when HERG K channels are blocked. It is seen that this parameter of the Thalassiolin B molecule does not meet the required conditions. The next parameter is QPPCaco, which is Caco-2 cell permeability at the gut-blood barrier for inactive transport. It is seen that the numerical value of this parameter is too little for this molecule 40 42 .

Anti-human colon cancer potentials of the Thalassiolin B
The MTT set is the best-known test for cell viability. The main purpose of this test is to evaluate the toxicity of compounds, drugs, or other supplements on the cell. Of course, it may also be mentioned in articles as a process for examining cell proliferation or counting 43 . MTT analysis can differentiate between living and dead cells by affecting intracellular organs. In this method, the cells, after being cultured in the laboratory, are "treated" with the desired substances to evaluate their toxicity. At the end of this test, for each concentration of the substance, the cell viability is determined. Although this method is primarily for water-soluble solutions and compounds, it is currently used for other compounds soluble in organic solvents and molecules. The behavior and rate of cell proliferation may increase or not change at all under the influence of hormones, growth factors, cytokines, and mitogens. Also, some drugs and cytotoxic toxic substances, such as anticancer drugs, may cause necrosis or apoptosis death of cells or slow down the rate of proliferation and growth or even loss of cell structure 44 46 . Proper analysis of the MTT test can evaluate many of these behaviors. The MTT analysis basis is based on mitochondrial activity. This activity is usually stable in living cells. Hence, any change in several active and living cells is linked to mitochondrial property. This examination is a colorimetric way based on the breakdown and reduction of yellow tetrazolium crystals by the succinate dehydrogenase, and the formation of insoluble purple crystals perform the final analysis. Unlike other methods, MTT analysis eliminates the cells washing and shrinking steps, which usually causes the loss of cells part and increases the work error. That is, all the steps of the experiment, from the cell culture, beginning to reading and analyzing the findings with a photometer, are done in a completely compact way and a "microplate". Hence the sensitivity, accuracy, and repeatability of the test are high 43 46 .
In the present study, the cytotoxicity of the Thalassiolin B and Cisplatin was explored by studying their interaction with normal HUVEC and EB, LS1034, and SW480 cell  The IC 50 of the Thalassiolin B against EB, LS1034, and SW480 were 483, 252, and 236 μg/mL, respectively. Thereby, the best cytotoxicity results and anti-human colon cancer potentials of our Thalassiolin B were observed in the case of the SW480 cell line Table 3 .

Antioxidant potentials of the Thalassiolin B
Maybe the antiproliferative effect of the Thalassiolin B is linked to its antioxidant activity Fig. 7 and Table 4 . Similar reports have indicated the antioxidant materials such as biological molecules reduce the volume of tumors by removing free radicals 47 . In detail, the high presence of free radicals in the normal cells make many mutations in their DNA and RNA, destroy their gene expression and then accelerate the proliferation and growth of abnormal cells or cancerous cells 48,49 .

Discussion
After the enzymes of the Thalassiolin B molecule were activated, ADME/T analysis was performed to theoretically calculate the effects and responses of the Thalassiolin B molecule on human metabolism in cells and tissues. Due to this analysis, many parameters have been obtained. The parameters obtained as a result of the calculations of the Thalassiolin B molecule are given in Table 3. The first parameter in this table is Solute Molecular Weight, which gives information about the molecular weight of the molecule being studied. Among all ADME/T parameters, another two important parameters are RuleOfFive and RuleOfThree. The RuleOfFive and RuleOfThree parameters are more important than any other parameter. The numerical value of these two parameters is expected to be zero. The RuleOfFive parameter, also known as Lipinski s, is Pfizer s fifth rule. The rules are: mol MW 500, QPlogP o/w 5, donorHB≤5, accptHB≤10. However, the RuleOfThree parameter is known as the three of Jorgensen s rule. The three rules are: QPlogS 5.7, QP PCaco 22 nm/s, #Primary Metabolites 7. If the numerical value of the RuleOfThree parameter is zero, this molecule can be used orally as a drug. The last and another important parameter is Jm, which is the predicted maximum transdermal trans- Fig. 4 The anti-human colon cancer properties of the Thalassiolin B against EB a , LS1034 b , and SW480 c cell lines.
A Pre-clinical Trial Study port rate, Kp MW S μg/cm 2 /hr . Kp and S are obtained from the aqueous solubility and skin permeability, QPlogKp and QPlogS. With this parameter, the theoretical estimates obtained by applying molecules that can be drugs to the skin are numerical values 42 .
The anticancer potential of therapeutic compounds against human colon cancer cell lines is immensely related to their antioxidant properties. Several earlier reports have uncovered that therapeutic compounds with strong antioxidant capacity significantly inhibit the growth of cancer cells by removing free radicals 8 . This is our earnest effort in exploiting biological molecule Thalassiolin B towards the adenocarcinoma studies and the corresponding results,  The different words indicate the significant difference (p ≤ 0.01) between several groups. Fig. 6 The cytotoxicity effects of the Thalassiolin B against Normal HUVEC cell line.
we believe, will open up a wing towards anticancer research in future. Now, turning our attention to investigate the bioactivity of the Thalassiolin B a concentration-dependent DPPH radical scavenging effect of molecule was observed against BHT as a reference. The interaction between the Thalassiolin B and DPPH might have occurred by transferring electrons and hydrogen ions 48 . The scavenging capacity of the Thalassiolin B and BHT at different concentrations, expressed in terms of percentage inhibition, has been shown in Fig. 6. In the antioxidant test, the IC 50 of butylated hydroxytoluene and Thalassiolin B were 175 and 133 μg/mL, respectively Table 4 . Biological molecules show higher antioxidant effects against free radicals formation into the living system 49 . The biological molecules have excellent redox properties and have a significant role in free radical deactivating 48,49 .

Conclusions
Biological activities of the Thalassiolin B molecule against enzymes were compared. Afterwards, a theoretical ADME/T analysis of this molecule was performed. The cal-culated parameters of the Thalassiolin B molecule were examined. The numerical values obtained from ADME/T calculations for the Thalassiolin B molecule show that some ADME/T parameters of the Thalassiolin B molecule do not meet the necessary conditions, theoretically, this molecule is not recommended to be used as a drug. But it should be kept in mind that experimental conditions differ from theoretical computational conditions. The numerical values of the parameters obtained from this study are used in the future in vivo and in vitro studies, providing a great deal for new drug candidate discovery.
The oncological part of the recent study was revealed the cytotoxicity and anti-human colon cancer properties of Thalassiolin B against EB, LS1034, and SW480 cell lines in the in vitro condition. The IC 50 of Thalassiolin B against EB, LS1034, and SW480 were 483, 252, and 236 μg/mL, respectively. The antioxidant properties of Thalassiolin B were determined against DPPH free radicals. The IC 50 of Thalassiolin B was 133 μg/mL. Maybe the anti-human colon cancer properties of Thalassiolin B are related to their antioxidant effects.