In Silico Studies, Biological Activities, and Anti-human Pancreatic Cancer Potential of 6-Hydroxy-4-methylcoumarin and 2,5-Dihydroxyacetophenone as Flavonoid Compounds

The collagenase enzyme is enzyme belonging to the hydrolase class breaks down the triple helix structure Abstract: Coronavirus is one of the RNA viruses with the largest genome; It is a group of viruses known to infect humans very little until the end of the 20th century, generally causing infection in animals (bird, cat, pig, mouse, horse, bat). It is the causative agent of 15-30% of seasonal lower and upper respiratory tract infections, and may rarely cause gastrointestinal and nervous system infections. We have obtained results for the collagenase and elastase enzymes were at the micromolar level. We obtained IC 50 results for the collagenase enzyme for 6-hydroxy-4-methylcoumarin 257.22 ± 34.07 µM and for 2,5-dihydroxyacetophenone 74.46 ± 8.61 µM. 6-Hydroxy-4-methylcoumarin and 2,5-dihydroxyacetophenone were considered good inhibitors for elastase enzyme. Additionally, these compounds significantly decreased human pancreatic cancer cell viability from low doses. In addition, 100 µM dose of all compounds caused significant reductions in human pancreatic cancer cell viability. IC 50 results (IC 50 : 10-50 µM) were better than control. In the otherwords, the docking results suggest that both compounds tend to have lower efficacy on the main protease targets of SARS-CoV-2 than standard compounds, (NL-1 and NL-2). The reason for this is that the standard compounds interact strongly and more frequently with the target proteins, and the surface areas they cover on the active surface are much larger than the small ligand molecules studied.

of collagen. Hydrolases provide hydrolysis reactions, that is, the destruction of molecules, with the help of H and OH ions of water. The enzyme, named after its substrate, is also known as matrix metalloprotease-1 or metallopeptidase-1. The weights of collagenases vary between 50-60 kDa, their cofactor is Zn metal. One group of proteases, which are extracellular proteolytic enzymes, need Zn 2 or Ca 2 ions in the bound state for their activation, while the other group is serine proteases containing reactive serine in their active sites 7,8 . Degradation of matrix proteins such as lanin, collagen and fibronectin by metalloproteases and serine proteases facilitate cell migration. Collagenase is one of these enzymes. These enzymes play a key role in physiological conditions such as normal structuring of tissues and systems, wound healing, tissue remodeling and normal development processes, and in pathological processes such as tumor cells spreading to surrounding tissues and disrupting their functions 9,10 . Elastases, an important enzyme belonging to the chymotrypsin family of serine proteases, are responsible for the fragmentation of extracellular matrix proteins such as elastin and collagen, which are responsible for skin elasticity and strength. The increase of elastase enzyme leads to deterioration of extracellular matrix components, changes in the structure of elastic fibers and consequently it leads to reduction of elastic properties of the skin and formation of wrinkle. In addition, the irregular and excessive release of elastase enzymes cause abnormal distortions in healthy tissues and this causes chronic wounds and inflammatory diseases. The development of elastase enzyme inhibitors due to these different effects is regarded as beneficial for protecting skin elasticity and controlling elastase-associated diseases 11,12 .
Pancreatic ductal adenocarcinoma is the fifth leading cause of cancer death in the Western world, with an overall 5-year survival rate of less than 1 and a median survival of 4 months after diagnosis. Histologically, cancer cells exhibit poorly differentiated ductal-like structures, often surrounded by an extensive desmoplastic reaction and infiltration by inflammatory cells 13,14 . The adjacent pancreatic parenchyma harbors sites of acinar cell degeneration and ductal cell proliferation. A high percentage of these cancers overexpress a number of growth factors and their receptors, including EGF, transforming growth factor TGF -α, CRIPTO, TGF-β1, epidermal growth factor re-ceptor, acidic FGF, basic fibroblast growth factor, and FGF5. Overexpression of these mitogenic growth factors may contribute to the bioaggression of pancreatic cancers and the abundant stroma formation that is characteristic of this malignancy 15,16 .
The aim of this study is to examine the potentials of anticollagenase, anti-elastase, anti-panceratic cancer, and anti-Coronavirus disease COVID-19 by using two important compounds and to detect them by molecular modeling.

Enzymes
0.05 mL was taken from the prepared sample solutions. On top of it, 0.05 mL of elastase enzyme 0.16 U/mL was added. Then, 0.9 mL of tris hydrochloride Tris-HCl buffer solution of 0.2 M pH 7.8 was added to the sample solutions. It was prepared by adding 0.2 M pH 7.8 0.9 mL Tris-HCl buffer solution to 0.1 mL of elastase enzyme solution as a control solution. The blank solution was prepared by adding 0.2 M pH 7.8 0.9 mL Tris-HCl buffer solution to 0.1 mL distilled water. Blank, control and sample solutions were incubated at 37 for 15 minutes. After incubation, 5 mM 0.05 mL N-Succinyl-Ala-Ala-Ala-p-nitroanilide STANA substrate was added to the blank, control and sample solutions and incubated at 37 for 30 minutes 17,18 .
The absorbance values of the sample and control solutions against the blank were read at 410 nm. In the study, the anti-elastase inhibition activity values of the samples prepared at different concentrations were calculated. Experiments were repeated 3 times and averaged. The inhibition values on elastase enzyme of eperezolide-like compounds synthesized for the first time were calculated. The IC 50 value the concentration required to inhibit 50 of the activity was calculated from the regression equation obtained from the linear segment of the curve drawn by applying the concentration to abscess, elastase enzyme inhibition data to the ordinate 19,20 .
Modified inhibitory effect on collagenase enzyme Thring et al. 2009 21 was determined spectrophotometrically using the method. 50 µL of the solution containing 0.8 U/ mL collagenase was taken, 50 µL of plant extracts and chemical substance solutions at different concentrations prepared on it were added 22 . This method was performed according to previous studies. The absorbance values of the sample solutions and control solution were read at 340 nm in the UV spectrophotometer against the blank. Experiments were repeated 2 times. The IC 50 value, which is the amount of substance required for the collagenase enzyme to have a 50 inhibition effect, was calculated with the regression equation obtained from the linear section of the curve drawn by applying the concentration to the abscess in the graph and the enzyme inhibition data to the ordi- In Silico Studies, Biological Activities, and Anti-human Pancreatic Cancer Potential of Flavonoids nate 23 .

Molecular docking of different targets Collagenase,
Elastase, and Major protease mpro of SARS-CoV-2 with the studied two molecules The docking of 6-hydroxy-4-methylcoumarin 1 and 2,5-dihydroxyacetophenone 2 as ligands with Collagenase, Elastase, and Major protease mpro of SARS-CoV-2 was analyzed using AutoDock Vina 24 . The ligands were drawn and optimized at DFT/B3LYP/6-311G* basis set using Gaussian 09 G09 25 . The crystal structures of targets, Collagenase pdb: 4AR1 , Elastase pdb: 1MCV and the main protease Mpro, pdbs: 6LU7 26 and 2GTB 27 structures were downloaded from the protein data bank http://www.rcsb.org were used for docking processes. Moreover, these target proteins were prepared and minimized until the root mean square deviation RMSD reaches the lower value of 0.05 kcal/mol Å 2 using Discovery Studio DS 3.5 28 . Define and edit binding site tool of DS was exerted to detect binding site of aforementioned targets against 1 and 2 ligands. AutoDock Vina 24 was employed to predict the conformations and binding interactions for the ligands. The molecular docking of all of the ligands with Collagenase, Elastase, and Major protease mpro of SARS-CoV-2 proteins were analyzed, where the respective targets were constant and the ligands were flexible. The best orientation for each complex was selected based on RMSD and the predicted binding energy of the ligands. Further, cluster analysis, based on RMSD values, was used and the most populated cluster with the lowest energy conformation was noted as an authenticated answer.

Cancer study 2.3.1 Replication of cells
In order to determine the cytotoxic activity of the compounds, human pancreatic cancer cell line was obtained from the American Type Culture Collection ATCC and used in the study. Cells were fed twice a week and cell flasks were incubated at 37 Thermo Forma II CO 2 Incubator, USA in a 5 CO 2 environment throughout the experimental period. Confused cells were removed with trypsin-EDTA solution and counted under the microscope after staining with 0.4 trypan blue. For experimental studies, 96-well plates were seeded with approximately 15 10 3 cells per well 29 .

Treatment with test compounds
1-100 µM concentrations of the compounds to be tested were added to the cell seeded wells, and then the plates were incubated for 24 hours at 37 in 5 CO 2 incubator. The possible effects of the applied compounds on cell viability at the end of the incubation were determined by the MTT method 30 .

MTT method
MTT solution at a concentration of 0.5 mg/mL was pre-pared for the analysis of viability levels in cells after compound administration. After the application, 50 µL of MTT solution was added to each well and incubated in a CO 2 incubator for 3 hours. After incubation, the solution in the wells was withdrawn and 100 µL of DMSO was added to them. The optical density of the cells in the wells was read in an ELISA plate reader Thermo MultiskanGo, USA at a wavelength of 570 nm. The absorbance values obtained from the control wells were averaged and this value was evaluated as 100 cell viability. The absorbance values obtained from the compound treated wells were proportioned to the control absorbance value and the percent viability values were calculated 31 .

Enzymes
Inhibition of elastase and collagenase enzymes have been one of the main targets in cosmetic industry for researches to find new antiwrinkle and skin-lightening compounds or extracts particularly with rich polyphenol contents as well as to prove their effectiveness 32 .
We have obtained results for the collagenase and elastase enzymes were at the micromolar level. We obtained IC 50 results for the collagenase enzyme for 6-hydroxy-4-methylcoumarin 257. 22

Molecular docking results
The compound 1 and 2 in this research exhibited affinity to Collagenase, Elastase, and Major protease mpro of SARS-CoV-2, with a minimum energy requirement. Among the 200 different conformers of each ligand tested for the inhibition of the related target, compound 2 displayed the potential for a high inhibition property based on the minimum energy of the ligand 7.35 kcal/mol, and 7.10 kcal/mol in Table 2 and Fig. 2 , to comply into the binding site of Collagenase and Elastase models. In vitro assay in this research has shown that administration of 6-hydroxy-4-methylcoumarin 1 and 2,5-dihydroxyacetophenone 2 , inhibited Collagenase and Elastase activities based on the standards, Phosphoramidon and N-Methoxysuccinyl -Ala-Ala-Pro-Val-chloromethyl ketone, respectively.
Besides these, compound 2 forms three hydrogen bonds with Glu430, Tyr428 and Gln462 in the binding site of collagenase; and also three hydrophobic interactions with His459 and Trp471amino acids of aforementioned target Fig. 3. In the meantime, The same compound has four H-bonds Cys191, Gly193, and Gly192 and one hydrophobic interaction with Val216 residue of elastase. These interactions of compound 2 against each target were represented in Fig. 4. Another one, compound 1 has three hydrogen bonds Gly463, Gln462 and Glu486 , six hydrophobic interactions Trp471, His459 and Trp471 with collagenase, Fig.  3 . For elastase protein, compound 1 occurs two H-bonds with Asp194, Ser195; one πlone pair bond with Ser195; and two hydrophobic interactions with His57 and Val216 residues of the target, as given in Fig. 4.
On the other side, the ligand molecule, 6-hydroxy-4-methylcoumarin 1 was found to interact with the   In Silico Studies, Biological Activities, and Anti-human Pancreatic Cancer Potential of Flavonoids binding site in the main protease Mpro, pdbs: 6LU7 and 2GTB models, requiring minimum energy of 5.83 kcal/ mol, and 6.32 kcal/mol, respectively Table 2 . The compound 1 exhibites the better activity than compound 2 against both main proteases. However, these small compounds have lower binding affinity than NL-1 and NL-2 as the standard compounds 8.65 kcal/mol, and 8.41 kcal/ mol in Table 2 .
In the otherwords, the docking results suggest that both compounds 1 and 2 tend to have lower efficacy on the main protease targets of SARS-CoV-2 than standard com-pounds, NL-1 and NL-2 . The reason for this is that the standard compounds interact strongly and more frequently with the target proteins, and the surface areas they cover on the active surface are much larger than the small ligand molecules studied. This situation is indicated visually in Figs. 5A and 5B, and numerically in Table 2.

Cancer results
The cytotoxic effect of test compounds on human npancreatic cancer cell lines is shown in Fig. 6. Compounds 6-hydroxy-4-methylcoumarin and 2,5-dihydroxyacetophe- Fig. 2 The optimized compound 1, 2, std-1 and std-2 at DFT/B3LYP/6-311G* level using Gaussian09. none significantly decreased human pancreatic cancer cell viability from low doses Fig. 6 . In addition, 100 µM dose of all compounds caused significant reductions in human pancreatic cancer cell viability Fig. 6 . In general, we can say that of the four tested compounds, 6-hydroxy-4-methylcoumarin and 2,5-dihydroxyacetophenone have cytotoxic effects in all cell types, and this effect is particularly strong in human pancreatic cancer cells.

Discussion
In this study, N-methoxysuccinyl -Ala-Ala-Pro-Valchloromethyl ketone was used as a standard for elastase. For 2,5-dihydroxyacetophenone compound, a good result was determined compared to the standard, but a poor result was calculated for 6-hydroxy-4-methylcoumarin compound compared to the standard. Inhibiting the activity of extracellular matrix-degrading ECM proteins such as elastases and collagenases may be a useful approach to prevent UV-induced skin changes and premature skin  In Silico Studies, Biological Activities, and Anti-human Pancreatic Cancer Potential of Flavonoids aging. Because reactive oxygen species ROS plays a key role in the activation of these enzymes, scavenging ROS by natural antioxidant compounds may be an option for inhibiting such skin-degrading enzymes. Phenolic molecules are an important class of natural antioxidants. They belong to various subclasses of secondary plant metabolites classified as stilbenes, flavonoids, phenolic acids and lignans and are ubiquitous in the plant kingdom 33 . Especially red and white grapes contain high amounts of phenolic acids and flavonoids such as catechin and gallic acid. Due to their chemical structure, polyphenols have strong antioxidant activities that scavenge a wide range of ROS such as superoxide radicals, hydroxyl radicals and Superoxide O 2 . In addition, polyphenols can inhibit the activity of proteolytic enzymes in vitro by acting as complexing or precipitating agents as noted in the literature. In particular, green tea polyphenols such as epigallocatechin gallate and catechin, which are widely used as ingredients in anti-aging skincare formulations, have been shown to exhibit moderate inhibitory effects against elastase and collagenase activity, possibly through non-covalent bonding 34 .
Pancreatic cancer is known to spread rapidly and is rarely detected in early stages. Not until quite advanced stages, no signs and symptoms are observed and in advanced stages, it is nearly impossible to remove the tumor. This type of cancer is known to be aggressive and migrate to different sections of the body quickly. If metastasis of pancreatic cancer could be stopped it would be possible to manage the tumor without searching for additional tumor spread and if this treatment could be combined with drug resistance decrease it could be used as a good treatment option for pancreas cancer 35,36 .

Conclusion
In this study, inhibition effects on both compounds, collagenase and elastase enzymes were determined, the results were obtained at micromolar level and micromolar acceptable. then the interactions between enzymes and compounds were examined with molecular docking and the results are similar and suitable to in vitro results, then Anti-COVID19 study was done with molecular docking program, for Anti-COVID the results are not as we wanted but not bad. We have studied the latest anticancer effects and obtained good results, so we can say that we can use these compounds in drug design in the future. The increase in elastase enzyme activity reveals the structural and functional changes of collagen and elastic fibers. This situation leads to the deterioration of the flexible structures of the skin, the formation of a rough texture and aging. Elastase inhibitors show anti-wrinkle activity that maintains skin elasticity. In addition, the imbalance between elastase and its natural inhibitors causes tissue damage and may cause lung and connective tissue diseases such as cystic fibrosis, asthma, pulmonary emphysema, respiratory distress syndrome. Therefore, it is thought that with the development of elastase-specific inhibitors, it will be possible to control elastase-related diseases.

Data Availability Statement
Data that support study findings are available with the corresponding author upon reasonable request.

Authors' Contributions
All authors have had a same role in preparing, designing, doing experiments, analyzing, writing, and submitting the recent manuscript.

Conflict of Interest
There isn t any conflict of Interest. Fig. 6 Investigation of the effects of these compounds on cancer tissue at different concentrations and their percentages in graphic form.

Supporting Information
This material is available free of charge via the Internet at doi: 10.5650/jos.ess22021