In silico and in vitro elastase inhibition assessment assays of rosmarinic acid natural product from Rosmarinus officinalis Linn

ABSTRACT The use of various herbs and their compounds has been a strategy widely used in the fight against various human diseases. For example, rosmarinic acid, a bioactive phenolic compound commonly found in Rosemary plants (Rosmarinus officinalis Labiatae), has multiple therapeutic benefits in different diseases, such as cancer. Therefore, the study aimed to evaluate in silico and in vitro the inhibition potential of the enzyme Elastase from the porcine pancreas by rosmarinic acid isolated from the plant species R. officinalis Linn. Through Molecular Docking, the mechanism of action was investigated. In addition, rosmarinic acid presented a range of 5–60 µg/mL and significantly inhibited Elastase. At 60 µg/mL, there was an inhibition of 55% on the enzymatic activity. The results demonstrate the inhibition of Elastase by rosmarinic acid, which can lead to the development of new enzyme inhibitors that can be an inspiration for developing various drugs, including anticancer drugs. Graphical Abstract

the rosmarinic acid (1) anticancer potential has been investigated nowadays, and several mechanisms of action have been proposed for its anticancer potential.for example, it impedes the development and growth of HL-60 promyelocytic leukemia cells (Saiko et al. 2015) and impairs tumor formation and evolution in mice with colon cancer (Venkatachalam et al. 2016).furthermore, it blocked a human ovarian cancer cell line, disrupting its cell cycle (tai et al. 2012), and ultimately prevented the generation of tumor cells in the skin of mice (Sharmila and Manoharan 2012).
one of the ways to understand the action of rosmarinic acid (1) on cancer is to understand the inhibition process of the enzyme elastase.elastases are a group of serine proteases involved in the degradation of fibronectin, cross-linked elastin, collagen, and other proteins.the human genome encodes six elastases secreted by the pancreas, neutrophils, macrophages, keratinocytes, and fibroblasts.the uncontrolled elastase activity is involved in inflammation diseases, delayed wound healing, and decreased skin elasticity (Siedle et al. 2001(Siedle et al. , 2007;;Mohanka et al. 2012). in addition, elastase can degrade a wide variety of functionally and structurally essential molecules in human tissues in addition to elastin.Although enzymes can have a salutary effect on renewing normal tissue and fighting infection, their excessive release (especially under conditions that compromise the function of their regulatory inhibitors) can lead to tissue damage in a broad spectrum of diseases (Janoff et al. 1979).pancreatic elastase is found in animal and human tissue (Largman et al. 1976).it is unique among animal tissue proteolytic enzymes that rapidly hydrolyze the scleroprotein elastin due to its unique specificity in degrading elastin.tests involving porcine pancreatic elastase and the α1-antitrypsin inhibitor have shown that its deficiency has been implicated in cancer.therefore, looking for compounds directly inhibiting the enzyme elastase is essential in screening possible drugs for various diseases such as cancer (Aronchik et al. 2010;Cui et al. 2021).
Although there is a wide range of research on elastase enzyme inhibitor compounds, it is still vital to study and discover new inhibitors due to existing enzymes' low efficacy and side effects.(Jakimiuk et al. 2021).thus, rosmarinic acid (1) is a potential inhibitor agent since, at a concentration of 25 µM, it reduced the elastase enzyme's activity by 80% (Karatoprak et al. 2020).furthermore, in in vitro induced changes in elastase, rA at a concentration of 100 µM inhibited the enzyme, altogether abolishing the changes (rama Devi et al. 2016).thus, current evidence encourages the exploration of rosmarinic acid (1) as a promising therapeutic agent against several disorders in the human body (Nadeem et al. 2019).previous studies indicate that rosmarinic acid (1) can act differently and exert innovative biological and pharmacological activities.therefore, a deep analysis of its characteristics and mechanism of action regarding its inhibition activity is necessary.in this work, the elastase inhibition activity from the porcine pancreas in vitro was evaluated, and its action mechanism in silico was investigated.

Isolation and in vitro elastase inhibition assays of the rosmarinic acid (1)
Maceration for seven days using water/acetic acid (85:15 v/v) of R. officinalis powered (200 g) followed by filtration and pH adjustment furnished the residue (27 g), which was fractionated by Sephadex LH-20, resulting in the isolation of rosmarinic acid (1) natural product.results of the NMr analysis (Supplementary data) and literature data confirmed the structure of the compound (Murata et al. 2010).Natural products provide unlimited opportunities for new protease inhibitors.plants produce a tremendous variety of chemical compounds to protect against herbivores and insects, and many of these are serine protease inhibitors (Wink 1988;Cuccioloni et al. 2009;Newman and Cragg 2016;Jakimiuk et al. 2021).polyphenols as inhibitors of serine proteases, including trypsin and elastase, are described by various authors (Wittenauer et al. 2015;Jakimiuk et al. 2021).
proteases, also called peptidases, proteolytic enzymes, or proteinases, catalyze the hydrolysis of peptide bonds in proteins and peptides.proteases can be classified as serine, cysteine, metallo, threonine and aspartic protease, depending on the nature of the amino acid residue or cofactor at the active site involved in the mechanism of enzyme catalysis (rawlings and Barrett 1993).Serine proteases such as elastase are the most widely studied proteolytic enzymes (Muri et al. 2005).therefore, looking for compounds directly inhibiting the enzyme elastase is essential in screening possible drugs for various diseases such as cancer.
thus, the elastase inhibition assay showed that the Concentration of rosmarinic acid (1) in the range of 5-60 µg/mL (figure S2) produced significant inhibition on elastase.At 60 µg/mL, there was an inhibition of 55% on the enzymatic activity.therefore, these results open up great perspectives in studying this compound regarding the potential elastase inhibition, and further studies are needed to understand its mechanism of action fully.

In silico elastase inhibition mechanism
table S1 shows the rMSD values for each function, diameter, and configuration.the lowest rMSD value for this data set was 0.9738 Å in the ChempLp score function within a 5 Å cavity of the elastin, having a flexible Ser195 residue along HoH281 structural water (Namba 2008).thus, this position very similar to the original oNo-6818 ligand from the .pdbfile.therefore, rosmarinic acid (1) has a better place and interaction with elastin.As this position is in the lowest rMSD value, the redocking was done and shown in figure S4.
in figure S4, the oNo-6818 original ligand interacts with hydrogen bonding with the following residues: Cys191, Gln192, Gly193, Asp194, Ser195, Ser214, Val216, and HoH281 structural water.it is worth describing that a covalent bonding bonded the crystallized ligands with elastase found in the .pdbfile in the Ser195 residue.the basic topological pattern of the ligand is to be C-C-o, and the carbons in this pattern are not aromatic.Also, the simulation results showed that the carbonyl group was relatively near to the Ser195 residue (~0,45nM) (Donarska et al. 2021).According to those data analyses, the GoLD software returned more than 100 positions from molecular docking simulation considering the optimized position of the rosmarinic acid (1).from those groups of positions, we selected the one with more hydrogen bonding and cation-π interactions in the residues that were similar to the oNo-6818 original ligand, which was: Cys191, Gly193, Gln192, Ser195, and Arg217.thus, in this condition, the n2 position was selected along with other optimal data, such as the lowest Gibbs energy ( . ) 'G u 112 8 10 3 1 kcalmol and highest fitness score (85.4261).figure S5 shows those interactions of the rosmarinic acid (1) and oNo-6818 molecules over the elastin protein.
After selecting the n2 position of the rosmarinic acid (1) due to the similarity of the interaction of the oNo-6818 original molecule from the .pdbfile, figure S6 shows the electrostatic surface potential of the complexed oNo-6818 molecule in comparison with rosmarinic acid (1) over elastin protein.
Compared with the oNo-6818 original ligand, the rosmarinic acid (1) molecule can interact in the same residue of the enzyme: Cys191, Gly193, Gln192, Ser195, and Arg217.Since the pancreatic porcine elastase is a protease that hydrolyzes peptides and proteins at bonds adjacent to the enzyme's neutral amino acid residues, the rosmarinic acid (1) will inhibit the protease in those residues.the rosmarinic acid (1) molecule had interacted directly with the -NH 2 groups and their neighbor atoms from the foretold residues of the protease.this molecule can inhibit the pancreatic porcine elastase by mostly hydrogen bonding over the -NH 2 and other polar groups, such as -C = o lateral bonding with -oH groups from the catechol group of the rosmarinic acid (1).As said before, the polar interactions can penetrate the elastase complex and inhibit it by occlusion of the catalytic site of the protease.the coupling of the rosmarinic acid (1), compared to the oNo-6818 ligand in the elastin protein, shows that rosmarinic acid (1) can go throughout a positive cavity of the active site of elastin (odagaki et al. 2001). in other words, the interactions of the rosmarinic acid (1) can potentially inhibit the active site of the elastin protein.However, in vivo and in vitro studies are necessary for an unequivocal description of the mechanism of action of this natural product.

Conclusion
rosmarinic acid (1) showed significant inhibition on elastase at 60 µg/mL.from the molecular docking results, it was possible to suggest that rosmarinic acid (1) can inhibit the active site of the elastin protein compared to the oNo-6818 ligand original