key: cord-0737146-trlezm8m
authors: Prastiwi, Rini; Elya, Berna; Hanafi, Muhammad; Saurisari, Rani; Desmiaty, Yesi; Dewanti, Ema; Herowati, Rina
title: The Chemical Constituents of Sterculia comosa (wall) Roxb Woods for Arginase Inhibitory, antioxidant Activity, and Molecular Docking against SARS CoV-2 Protein
date: 2022-01-19
journal: Heliyon
DOI: 10.1016/j.heliyon.2022.e08798
sha: 4397fc6412f0324f5d68464229b6c726b7719efe
doc_id: 737146
cord_uid: trlezm8m

Flavonoids and phenols have an arginase inhibitory and antioxidant activity. The Sterculia genus has phenols and flavonoids content. This study aimed to investigate the arginase inhibitory and antioxidant activity of the chemical constituent of Sterculia comosa (wall) Roxb and also their binding affinities to arginase. The most active extract was methanol extract. This active extract was determined for its arginase inhibitory and antioxidant activity, determined the total phenols and total flavonoids, and identified chemical compound. The methanol extract has IC(50) 2.787 μg/ml for arginase inhibitory activity and IC(50) 4,199 μg/ml for DPPH scavenging activity. The total phenols 723.61 mg GAE/gr, total flavonoids content 28.96 mg QE/gr extract. The chemical constituent: KC4.4.6 ((-)-2-(E)-caffeoyl-D-glyceric acid) and KC4.4.5.1 (trans-isoferulic acid) have an arginase inhibitory activity KC4.4.6: 98,03 μg/ml and KC4.4.5.1: 292,58 μg/ml. Antioxidant activity with DPPH methods KC4.4.6: 48,77 μg/ml and KC4.4.5.1: 88,08 μg/ml. Antioxidant by FRAP methods KC4.4.6: 16,4 FeEAC mol/g and KC4.4.5.1: 15,79 FeEAC mol/g. The isolate trans-isoferulic acid predicted has good interaction to arginase. Isolate KC4.4.6. Predicted has good interaction to PLPro of SARS CoV-2 PLpro. However, both isolates did not show good interaction to 3CLPro, nsp12, and Spike protein of SARS CoV-2.

The rapid and progressive spread of the SARS-CoV-2 virus pandemic caused by the coronavirus has severely affected thousands of people's health, the world's health system and has had a significant impact on the global economy. Characteristics of SARS-CoV-2 with influenza are the higher transmission rates and the greater risk of death from COVID-19, mainly due to Acute Respiratory Distress Syndrome (ARDS). The leading cause of death from COVID-19 in the elderly and those with compromised immune systems is respiratory failure.

Several patients exhibited cardiovascular-related pathologies, including Congestive Heart Failure (CHF) and cardio-respiratory medullary cardiac dysfunction. Cardiovascular complications and focus of ACE2 as a co-receptor for SARS-CoV-2 are essential in this regard. This viral infection depends on the entry of the virus through the host to replicate. Coronaviruses such as SARS-CoV-2 and SARS-CoV-1 use the host protein angiotensin-converting enzyme-2 as a co-receptor to gain access to the lungs and brain (Discovery, 2020) .

In coronavirus disease, the elderly and those with disorders such as hypertension, chronic obstructive pulmonary disease, diabetes, and cardiovascular disease are more likely to experience this virus adverse effects and become critical. They can rapidly develop acute respiratory distress syndrome, septic shock, metabolic acidosis, and coagulation dysfunction, possibly leading to death. Arginine supplementation can play a role in such a scenario, given the possibility of being in a state of deficiency (Mahmoud et al. 2020) . Nitric Oxide (NO) is an essential molecule in regulating intercellular signaling and is involved in various processes, including regulating endothelial function. NO has antimicrobial activity, including against bacteria, protozoa, and some viruses. NO produced by an enzyme that catalyzes L-arginine oxidation to NO and L-citrulline, namely NOS (Nitric Oxide Synthase). Aubrey et al.'s (2005) research gave the result that NO specifically inhibited the SARS CoV-1 replication cycle, especially at the beginning. NO production by iNOS produces antiviral effects (Aubrey-chimene et al., 2005) . In the COVID-19 pandemic, neonatal patients' treatment with inhaled NO was beneficial (Lu et al., 2020) .

Coronavirus and influenza are pandemic viruses that can cause lung injury and death due to ARDS. Viral infection can cause a "cytokine storm" that leads to pulmonary capillary endothelial cell activation, neutrophil infiltration, and increased oxidative stress. ARDS, characterized by severe J o u r n a l P r e -p r o o f hypoxemia, is usually accompanied by uncontrolled inflammation, oxidative injury, and damage to the alveolar-capillary barrier. Increased oxidative stress is a major cause of lung injury, including acute lung injury (ALI) and ARDS, two clinical manifestations of acute respiratory failure with high morbidity and mortality rates (Discovery, 2020) . Efforts to find effective compounds in inhibiting infection by the coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2) are still needed. Molecular docking analysis needed to find effective compounds against various target proteins for the treatment of coronavirus infection. This virus encodes a replicase complex (ORF1ab). They expressed in the form of the polyprotein (pp), which synthesizes nonstructural protein (nsp, nonstructural protein) and 4 structural proteins: spike protein (S), envelope (E), membrane (M), and nucleocapsid (N), during the proteolytic process.

The main protease, 3CL protease (3CLpro), is a key enzyme in processing pp1a and pp1b polyproteins. ORF1a and ORF1b terminated by papain-like protease (PLpro, nsp3) and 3C-like protease (3CLpro, nsp5) to produce nsp. The 3CLpro protein has an important function and is considered an active target for antiviral drugs. PLpro is an enzyme indispensable for viral replication and infection and is an essential target for coronavirus inhibitors. (Yu et al., 2020) .

Recent studies have shown that 2019-nCoV uses angiotensin-converting enzyme-2 as an entry receptor for entry into host cells. Protein S, a type 1 glycoprotein on the virus's surface, plays a vital role during virus entry into the host cell. Protein S helps the virus bind to host acceptors. This protein S from CoV2 has a strong affinity for human angiotensin-converting enzyme-2 (ACE-2).

Compounds that can inhibit this protein will potentially inhibit the entry of the CoV-2 virus into the human body. (Yu et al., 2020; Yulong et al., 2020; Zu et al., 2020) . the inhibitory effect on arginase enzymes such as methanol extract from Scutellaria indica and piceatannol-3'-β-Dglucopyranoside compounds from rhubarb extract (Kim et al. 2013) . Several studies on the inhibition of arginase activity in some plants have resulted that (A. Najid et al., 2018) Bordage et al., 2013) . Administration of antioxidants (vitamin C) shows reduce oxidative stress, which causes acute inflammation and lung injury. Antioxidants' administration also reduces the risk of viral infections and improves symptoms ( Discovery, 2020) .

J o u r n a l P r e -p r o o f 

The extraction was done by the maceration method using n-hexane, ethyl acetate, and methanol solvent. The extract dried in a vacuum of the rotary evaporator at temperature of 50 °C, continued in waterbath at 50 °C. The three extract tested the arginase inhibitor and antioxidant Activity. The active extract determine the flavonoids total and phenols total.

For initial screening of the extract's inhibitory activity (the final concentration of the test sample in the reaction is 100 μg/ml). The concentration of stock made is 450 µg/ml. Ten ( 

The extracts (20 µg/ml) of sample in methanol were reacted with 180 ml of 150 µmol/l DPPH (2,2-diphenyl-1-picrylhydrazyl) in methanol solution at room temperature. For a control, methanol used to replace the sample. The incubated at room temperature for 40 min in the dark place. The absorbance was measured at 517 nm. The positive control was quercetine. The antioxidant capacity was calculated using the following:

Equation 1 The procedure according to Bobo Garcia (Bobo-garcía et al. 2015) .

The FRAP method, for sample preparation, was carried out by five (5) FeEAC= △ x

x D x 1 x 10 5

The formula, FeEAC was the equality of ferric ions with antioxidant activity (µmol / g), which ∆A = absorbance of samples that have been reduced by blank, GRAD (M -1 ) was the gradient of the AFS calibration graph, Av = total volume for the test (300μl) , Spv = sample volume (30μl), Cext = concentration of sample stock, weight (gram) in volume (g / l), D = dilution factor for sample before analysis (D = 1 if sample was not diluted). GRAD (gradient) was determined from the calibration curve on AFS.

J o u r n a l P r e -p r o o f 2.5.

TPC expressed as mg Gallic acid equivalents per gram of dried extract (mg GAE/g Extract). A total of 20 µl extract added with 100 µl of Folin-C Reagent (1:10), treated for 60 seconds, and then allowed to stand for 4 min. Added with 80 µl of the solution of 7.5% sodium carbonate (Na2CO3) in water, shake for 60 seconds. This mixture is incubated at room temperature in a dark place for 2 h. Read at 600 nm. The concentration of extract in the sample made at 100 μg/ml. The concentration of a stock solution made was 1000 µg/ml. Blangko was a sample replaced with methanol. The treatment was the same as the sample. Determining the total phenol content using gallic acid standards, total phenol was calculated as gallic acid's equivalence 

The isolation of chemical constituents from Sterculia comosa shown in figure 1.

Ligand structures were drawn by MarvinSketch 17.8. The 3d structure of each ligand was generated using VegaZZ, charge addition and energy minimization were done under the Vina force field in VegaZZ and saved in mol2 format.

The 3D structure of arginase was obtained from RCSB with PDB id 4HZE (Zandt et al. 2013 ). The 3D structures of structural and non-structural protein of SARS-CoV-2 i.e. PLpro, 3CLpro, nsp12, and Spike obtained from RCSB with PDB id: 7CJM, 7JU7, 6NUS, and 6MOJ, J o u r n a l P r e -p r o o f respectively. Protein structure preparation done using AutoDock Tools. All non-standard residues and most water molecules were cleaned by removed from the initial structure except those involved in ligand-protein interaction. Then, all missing hydrogens and Kollman charges added to the system, and the prepared protein receptor then saved as pdbqt format (Ravi & Krishnan 2016) .

Docking validation was done through redocking of native ligand to its respective protein. docked potent agents and the targets were analyzed using PLIP (Salentin et al. 2015) .

The screening arginase activity showed that an active extract was methanol extract. The study of arginase activity performed on methanol extract obtained IC50 2.787 µg/ml while the value of IC50

for nor-NOHA as a positive control was 3.773 µg/ml. The result shown in table 1. the arginase inhibitor activity shown in figure 2.

The antioxidant activity using DPPH method obtained The IC50 FeEAC (mol/g).

Quercetin levels calculated as total flavonoid levels in the sample. 

Activity tests carried out on compounds that have been isolated show that the compound with the highest enzyme inhibitor activity is KM3.9.1 with an IC50 value of 59.31 µg/ml. NO is an essential molecule in regulating intercellular signaling and is involved in various processes, including regulating endothelial function. NO has antimicrobial activity, including against bacteria, protozoa, and some viruses. The result of Arginase Inhibitory Activity by Isolates show on table 7.

The antioxidant activity test results using the DPPH method showed that the compound with the highest antioxidant Activity was KC4.4.6 with an IC50 value of 48.77 µg/ ml. The antioxidant activity test results using the DPPH method showed that the most active isolate was KC4.4.6. This compound found in Sterculia comosa wood. In the form of wood extract, Sterculia comosa has better Activity than Sterculia macrophylla. The presence of a hydroxyl group in the KC4.4.6 compound contributed to increased antioxidant and arginase inhibitory activity. In the pathological condition, when arginase activity increases, eNOS becomes uncoupling, uncoupling eNOS will produce more free radicals, namely O2-and ONOO-. The use of the DPPH method for which the radical scavenging mechanism will be suitable. So that free radicals produced from The isolate results showed that the arginase inhibitory and antioxidant activity were smaller than the fraction, due to the possibility of synergism, complementary between compounds in the fraction that contribute to activity to increase the inhibitory activity of arginase and antioxidant enzymes.

Validation of the docking method carried out on a complex of arginase with the native ligand. The result determine as the RMSD value, which states the difference in the distance between the atoms of the redocking native ligand and the X-ray crystallography results. The validation result showed that the RMSD was valued 1.342 Ǻ, so the docking method was valid.

The overlay of redocked and crystallographic conformation of the native ligand can observed in figure 7.

Nitric oxide (NO) is an essential intercellular signaling molecule that inhibits some viral infections. NO reported to inhibit RNA synthesis and inhibit the SARS CoV replication cycle J o u r n a l P r e -p r o o f (Åkerström et al. 2005) . Arginase is an enzyme that will break down arginine as the starting material for NO. Arginase inhibition will increase NO levels.

The native ligand of 4HZE, i.e. [(5R)-5-amino-5-carboxy-7-(piperidine-1-yl)heptyl]

(trihydroxy)borate(1-), interacted to Asp147, Asn149, Ser156, His160, Gly161, Asp202 amino acid residues of 2-arginase by hydrogen bond. In contrast, non-hydrogen bond interactions observed to His120, His145, Asp200, as well as Asp202 (Zandt et al. 2013) . Nor-NOHA actively inhibited 2-arginase and was used as a control compound (Tenu et al. 1999) . Trans-isoferulic acid showed lower binding energy than native ligand and nor-NOHA. However, the binding energy of KC4.4.6 was higher than the native ligand ( Table 8 ).

The results of molecular docking of the ligands against SARS-CoV-2 targets proteins are presented in tables 9,10 and 11. KC4.4.6. predicted have the best interaction with PLpro. However, both trans-isoferulic acid and KC4.4.6 did not show good interaction to 3CLPro, nsp12, and Spike (table 10 and table11).

The interaction of native ligand, nor-NOHA, and isoferulic acid to the amino acid residues of arginase can observed in figure 8.

Arginine metabolism plays an important role in vascular function. The enzyme associated with the metabolism of arginine was arginase. This enzyme also an effect on vascular function. Our study showed that sterculia comosa has an arginase inhibitory activity. This extract also have a high of total phenols and total flavonoids and this result as the same as our previous study.

In the recent study the stem bark of Caesalpinia turtuosa has the arginase activity with the IC50 11.58 µg/ml for methanol extract and 33.81 µg/ml for ethyl acetate extract; this result was the same as our study. (Najid et al. 2018) patients' treatment with inhaled NO was beneficial (Lu et al., 2020) . NO plays an important role in maintaining normal endothelial function. The reduced availability of NO causes cardiovascular, neurological, cancer, and respiratory disorders. The reduction in NO production occurs in line with the increased reactive oxygen species (Khaled, 2016) . Based on Chandra (2011) , an increase in ONOO-and H2O2 oxidative stress will cause an increase in PKCα / β and then activate RhoA / Rho kinase (ROCK) and cause arginase activity to be excess. Based on this, the use of an antioxidant can function in inhibiting arginase activity.

Nor-NOHA as a control as an arginase inhibitor. The results of molecular docking to arginase revealed that Nor-NOHA formed 2 hydrogen bonds and 3 non-hydrogen bonds the same as the native ligand. Trans-isoferulic acid was the ligand with the lowest binding energy to arginase.

It formed several identical hydrogen bonding and non-hydrogen bond interactions with the key active site amino acids compared to the native ligand. The phenolic group of iso-ferulic acid plays an important role in the interaction to Ser156, Asn158, and His160, as the hydrogen bond acceptor.

The carboxylic acid of iso-ferulic acid act as a charge center to form electrostatic interaction to His120, His145, and His160 (figure 2). KC.4.4.6 also formed some similar interactions as the native ligand. However, the higher energy binding probably due to the high distance of some of the hydrogen bonds. Dihydroxy-propyl substituent to carboxylic group of iso-ferulic acid reduced carboxylic acid interaction to the key amino acid residues, producing lower binding energy.

Native ligand (5-amino-2-methyl-N-[(1R)-1-naphthalen- 

The chemical constituent of extract Sterculia comosa (Wall) Roxb has arginase inhibitory activity and antioxidant activity. The isolate trans-isoferulic acid predicted have good interaction to arginase. Isolate KC4.4.6. ((-)-2-(E)-caffeoyl-D-glyceric acid) was predicted to have good interaction to PLPro of SARS CoV-2 PLpro. However, both isolates did not show good interaction to 3CLPro, nsp12, and Spike protein of SARS CoV-2.

This research supports The Ministry of Research, Technology, and Higher Education, The

Republic of Indonesia, for the Research Foundation through the PDUPT Grant. 

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