the potential role of rosmarinic acid and sinensetin as α

DOI:10.21776/ub.jpacr.2019.008.001.460J.PureApp.Chem.Res.,2019,8(1),73-7910April2019

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Thejournalhomepagewww.jpacr.ub.ac.idp-ISSN:2302–4690|e-ISSN:2541–0733 Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution-NonCommercial4.0Internationalwhichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.(http://creativecommons.org/licenses/by-nc/4.0/)

The Potential Role of Rosmarinic Acid and Sinensetin as α-Amylase Inhibitor: In Silico Study

Hazna Noor Meidinna,1 Fatchiyah1,2*

1Department of Biology, Faculty of Mathematics and Sciences, Brawijaya University, Indonesia

2Research Center of Smart Molecule and Natural Genetic Resources, Brawijaya University, Indonesia

*Corresponding email: [email protected]

Received 4 March 2019; Accepted 10 April 2019

ABSTRACT The study of a natural compound as α-amylase inhibitor has been a concern since the synthetic drugs for the management of type 2 diabetes mellitus have several side effects. The present study was carried out to predict the ability of rosmarinic acid and sinensetin as a human α-amylase inhibitor by in silico study. All of the prepared 3D structures were used in the molecular docking by using Hex 8.0.0. The visualization of the molecular interactions of those compounds with human salivary α-amylase or human pancreatic α-amylase was established in the Discovery Studio Client 4.1 software. The result of this study determined that rosmarinic acid and sinensetin bound to the A domain of human pancreatic α-amylase and human salivary α-amylase. The rosmarinic acid-human salivary α-amylase complex was observed to possess a high number of hydrogen bonds compared to sinensetin-human salivary α-amylase complex. The similar result was observed in the comparison of rosmarinic acid-human pancreatic α-amylase complex and sinensetin-human pancreatic α-amylase complex. The rosmarinic acid was able to bind the Glu233 of human pancreatic α-amylase. These data suggest rosmarinic acid as a potential inhibitor of human salivary α-amylase and human pancreatic α-amylase. Further experimental evidence is needed to confirm this observation. Keyword: α-amylase inhibitor, in silico, rosmarinic acid, sinensetin

INTRODUCTION

The digestion of starch in a human occurs in several stages. Alpha-amylase is the main enzyme in human that is responsible for the breakdown of starch to more simple sugars (dextrin, maltotriose, maltose, and glucose) [1]. The parotid glands and the pancreas are the predominant sources of α-amylase. Salivary α-amylase is the most abundant enzyme in human saliva. Pancreatic α-amylase is synthesized in the pancreas and secreted into the small intestine. Both of them have a role in the initial break down of starch to oligosaccharides through its hydrolytic activity [2]. It is known that the high intake of simple carbohydrates may contribute to weight gain and increased postprandial glycemia [3].

The disorders of carbohydrate uptake may cause severe health problems. One of them is diabetes mellitus. One of the approaches to overcome metabolic diseases is the use of drug targeting carbohydrate digestion and absorption [4]. Acarbose is the drug functioned to lower the post-prandial glucose level through the prolong of overall carbohydrate digestion time. Acarbose is a pseudo-tetrasaccharide drug used as a second or third line agent in the treatment of type 2 diabetes mellitus. This drug acts as an inhibitor of complex starches to

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oligosaccharides hydrolysis in the lumen of the small intestine. Due to the side effects of the drug, the use of natural compounds for effective alpha-amylase inhibitor becomes a concern among researchers [4,5].

Rosmarinic acid and sinensetin that have been investigated in vitro are predicted to function as amylase inhibitors [6,7,8]. Rosmarinic acid is one of the polyphenolic substances occurring in many plant species [9,10] Sinensetin is found in citrus and other plant sources [7]. Both of them have physiological functions including antioxidant, anti-inflammation, and as immunomodulatory on specific tissues [9]. The main focus of this study determines the function of rosmarinic acid and sinensetin as a human α-amylase inhibitor by docking analysis. We predict that these compounds have the ability to bind the human α-amylase binding domain so that the starch carbohydrate digestion and glucose absorption will be slowed down. EXPERIMENT Chemicals and Instrumentation

The chemical structure of acarbose (CID:41774), rosmarinic acid (CID:5281792), and sinensetin (CID:145659) were retrieved from the database of PubChem. Protein Data Bank (PDB) was used as a database to get 3D structures of human salivary α-amylase (PDB ID:1SMD) and human pancreatic α-amylase (PDB ID:1HNY). Open Babel tool in PyRx Virtual screening software (version 0.8) was functioned to convert SDF file format to PDB format. Computational docking was conducted by using the molecular docking tools of Hex 8.0.0. The ligand-protein interactions were further visualized and analyzed by using Discovery Studio 4.1 software. Ligand and Protein Preparation

The 3D structure of acarbose, rosmarinic acid, and sinensetin were obtained in the form of SDF format from the database of NCBI. The energy form of those compounds was minimized and converted to PDB format by PyRx Virtual screening tool. Acarbose was used as a standard drug. There were two kinds of human α-amylase (salivary and pancreatic α-amylase), which share about 97% homology, used in this study. The water molecules incorporated in their 3D structures were removed. All of the prepared 3D structures were used in the molecular docking phase. Molecular Docking

Each of human α-amylase was docked to acarbose, rosmarinic acid, and sinensetin so that there would be six models of protein-ligand interactions. Docking was established by using the Hex 8.0.0 software. We used blind docking and default parameter of Hex 8.0.0. The molecular interactions of those compounds with human salivary α-amylase and human pancreatic α-amylase were further visualized in the Discovery Studio Client 4.1 software. RESULT AND DISCUSSION Analysis of Acarbose and Human Salivary α-amylase or Human Pancreatic α-amylase Interaction

The docking between acarbose and human salivary α-amylase was established. The ligand-protein interaction was shown by the binding site amino acid residue and the types of chemistry bond (Table 1). Six amino acid residues in A domain of human salivary α-amylase were able to interact with acarbose (Figure 1 A), namely Tyr2, Asn5, Thr5, Gln7, Gln8, and Arg252. The energy binding of the acarbose-human salivary α-amylase complex was -336.9


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kJ/mol. Those interactions occurred by the presence of hydrogen bond, whether conventional hydrogen bond or carbon-hydrogen bond. Acarbose did not bind the active site of human salivary α-amylase. But it is predicted that acarbose has a function as an amylase inhibitor which is likely to be stronger or the same as if it binds the active site.

The acarbose-human pancreatic α-amylase complex turned out to have less interaction compared to the acarbose-human salivary α-amylase complex (Table 2). According to the docking analysis in Figure 1 D, Trp59 was the only amino acid residue that bound to acarbose by establishing Pi-Donor hydrogen bond. The acarbose-human pancreatic α-amylase complex had the energy binding of -355.0 kJ/mol.

Other docking studies of acarbose-human pancreatic α-amylase complex observed Trp59 as one of the binding site amino acid residue [11]. It might be implied that this residue plays a significant role in the substrate binding site of α-amylase. The study of estimated IC50 values among acarbose, tetracycline, enalapril, and captopril was established. Acarbose was found to have the lowest IC50 values (0.0062 mM) in comparison to the three drugs [12].

Analysis of Rosmarinic Acid and Human Salivary α-amylase or Human Pancreatic α-amylase Interaction

Rosmarinic acid showed the activity of enzyme inhibitor and nuclease receptor ligand based on bioactivity prediction using molinspiration. This compound bound to the A domain of human salivary α-amylase. The analysis showed that rosmarinic acid bound to human salivary α-amylase at several sites, including Arg319, Thr376, Arg387, Trp388, Arg389, and Gln390. These binding sites in the rosmarinic acid-salivary α-amylase complex (Thr376, Arg387, Arg389, Gln390) were maintained by hydrogen bonds (Figure. 1 B, Table 1). The rosmarinic acid-human salivary amylase complex had the energy binding of -288.9 kJ/mol.

Interestingly, the interaction between rosmarinic acid and human pancreatic α-amylase occurred at amino acid residues in A and B domain, such as Tyr62, Gln63, Val107, Leu162, His299, and Glu233. These binding were maintained by hydrogen bonds as well. In addition to hydrogen bond, there were two residues, Tyr62 and Val107, which interacted with rosmarinic acid by hydrophobic interaction (Figure 1 E, Table 2). The rosmarinic acid-human pancreatic amylase complex had the energy binding of -296.8 kJ/mol.

The α-retaining double displacement is the generally accepted catalytic mechanism the α-amylase family. There are two catalytic residues in the active site of the amylase enzyme involved in the mechanism. The first residue is glutamic acid as the acid/base catalyst and the other one is aspartate as the nucleophile [13]. Structural and kinetic studies of human pancreatic amylase found that the side chain of Asp197 is the likely nucleophile in the catalytic mechanism of α-amylase. The side chains of Glu233 and Asp300 most likely involved in the catalytic process [14].

Glu233 may act as an acid/base catalyst in the hydrolysis of starch [14]. The ability of rosmarinic acid to interact with Glu233 indicated that the starch carbohydrate digestion might be retarded. Besides, histidine, arginine, and tyrosine play a role in positioning the correct orientation of the substrate into the active site of the α-amylase family. Proper orientation of the nucleophile and transition state stabilization are affected by these residues as well [13]. Normal substrate cleavage occurs between the -1 and +1 subsites [14]. The formation of a hydrogen bond between rosmarinic acid and His299 might cause the disorientation of the substrate. Tyr62 might affect the substrate and nucleophile orientation through hydrophobic interaction.


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Table 1. Interaction of Human Salivary α-amylase and Acarbose, Rosmarinic Acid or Sinensetin

Interaction Name Chemistry Bond Types

Acarbose-Human salivary α-amylase

A:ARG252: HH21-: LIG1:O

Hydrogen Bond Conventional Hydrogen Bond

A:ARG252:HH22 - :LIG1:O


:LIG1: H-: LIG1:O Hydrogen Bond Conventional Hydrogen Bond

:LIG1: H - A: TYR2:O Hydrogen Bond Conventional Hydrogen Bond

A:ASN5:CA - :LIG1:O Hydrogen Bond Carbon-Hydrogen Bond A:GLN8: CA-: LIG1:O Hydrogen Bond Carbon-Hydrogen Bond :LIG1: H - A: THR6:O Hydrogen Bond Carbon-Hydrogen Bond :LIG1: H - A: THR6:O Hydrogen Bond Carbon-Hydrogen Bond :LIG1: H - A:THR6: OG1

Hydrogen Bond Carbon-Hydrogen Bond

:LIG1: H - A: GLN7:O Hydrogen Bond Carbon-Hydrogen Bond :LIG1:H - :LIG1:O Hydrogen Bond Carbon Hydrogen Bond :LIG1: H - A:ASN5: OD1


:LIG1: H-: LIG1:O Hydrogen Bond Carbon-Hydrogen Bond

Rosmarinic acid-Human

salivary α-amylase

:LIG1: H – A:THR376: OG1


:LIG1: H - A: ARG387:O


:LIG1: H - A:GLN390: OE1


A:ARG389:HN - :LIG1 Hydrogen Bond Pi-Donor Hydrogen Bond

: LIG1:O – A: TRP388 Other Pi-Lone Pair : LIG1 - A: ARG389 Hydrophobic Pi-Alkyl : LIG1 - A: ARG319 Hydrophobic Pi-Alkyl

Sinensetin-Human salivary α-amylase

A: SER3HG-: LIG1:O Hydrogen Bond Conventional Hydrogen Bond

A:SER3:CB - :LIG1:O Hydrogen Bond Carbon Hydrogen Bond :LIG1: H – A:ASP402: OD2


:LIG1:H - :LIG1:O Hydrogen Bond Carbon Hydrogen Bond :LIG1:H - :LIG1:O Hydrogen Bond Carbon Hydrogen Bond :LIG1: H-: LIG1:O Hydrogen Bond Carbon-Hydrogen Bond

Note: Bold letters show the H-donor in hydrogen bond interaction, Lone Pair in Pi-lone pair interaction, Pi-Orbitals in Pi-Alkyl and Pi-Pi Stacked interaction, and Amide in Amide-Pi Stacked interaction


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Table 2. Interaction of Human Pancreatic α-amylase and Acarbose, Rosmarinic Acid or Sinensetin

Interaction Name Chemistry Bond Types

Acarbose-Human pancreatic α-amylase

:LIG1: H-: LIG1:O Hydrogen Bond Conventional Hydrogen Bond



:LIG1: H - A: TRP59

Hydrogen Bond Pi-Donor Hydrogen Bond

Rosmarinic acid Human pancreatic α-amylase –

A:GLN63: HE21-: LIG1:O


:LIG1: H – A: LEU162:O


:LIG1: H – A:HIS299: NE2


:LIG1: H – A:GLU233: OE1


A: TYR62-: LIG1 Hydrophobic Pi-Pi Stacked : LIG1 – A: VAL107

Hydrophobic Pi-Alkyl

Sinensetin-Human pancreatic α-amylase




A:ALA260:C,O;LYS261:N - :LIG1

Hydrophobic Amide-Pi Stacked

: LIG1 – A: ALA260

Hydrophobic Pi-Alkyl

Note: Bold letters show the H-donor in hydrogen bond interaction, Lone Pair in Pi-lone pair interaction, Pi-Orbitals in Pi-Alkyl and Pi-Pi Stacked interaction, and Amide in Amide-Pi Stacked interaction

Analysis of Sinensetin and Human Salivary α-amylase or Human Pancreatic α-amylase Interaction

There were three hydrogen bonds established between sinensetin and salivary α-amylase. The interacted residues were Ser3 and Asp402 (Figure 1C, Table 1). The sinensetin-human salivary amylase complex had the energy binding of -291.2 kJ/mol. No hydrogen bond existed in the sinensetin-pancreatic α-amylase complex (Table 2). The interactions happened in the complex were hydrophobic interaction, involving Ala260 and Lys261 (Figure 1 F). The sinensetin-human pancreatic amylase complex had the energy binding of -326.3 kJ/mol.

The active site residues of human salivary α-amylase are Asp197, Glu233, and Asp300 in the A domain. The human pancreatic α-amylase has three amino acid residues as active sites as well. The difference between human salivary α-amylase and human salivary α-amylase is


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located at the 14 amino acid residues mostly in the A domain. The residues caused the more negative charge of salivary α-amylase than pancreatic α-amylase [15,16].

Figure 1. Interaction of Acarbose, Rosmarinic Acid or Sinensetin and Human α-amylase. 1. The 3D molecular interaction. 2. The 2D molecular interaction

Flavonoids belong to a set of natural substances with different benzopyran structures [17].

Flavonoids demonstrated the inhibitory activities with the potential of inhibition related to a number of hydroxyl groups in the molecule of the compound [18]. Several in silico studies of the amylase inhibitory activity of flavonoids have been established. Butein was proved to bind the human pancreatic α-amylase with Asp197, Glu233 and Asp 300 as the potential binding site. Tristin was observed to have Glu233 of human pancreatic α-amylase as the potential binding site [17]. Although it is a flavonoid compound, sinensetin might not show significant human salivary α-amylase inhibition activities since there were only three hydrogen bonds and the involving amino acid residues were not located around the subsites of the active site. The in vivo and in vitro pharmacological consequences of human α-amylase inhibition by sinensetin need to be further characterized. CONCLUSION

The interaction between rosmarinic acid and human pancreatic α-amylase or human salivary α-amylase implies that rosmarinic acid has a higher potential ability as α-amylase


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inhibitor compared to sinensetin. Further investigations on both compounds and in vivo studies are necessary to develop a potential human α-amylase inhibitor for the prevention and treatment of diabetes.

ACKNOWLEDGMENT

We are sending our regards to our research group, SMONAGENES UB, for supporting this research. REFERENCES [1] Butterworth, P. J., Warren, F. J., Ellis, P. R., Starch, 2011, 63, 395-405. [2] Blanco, A., Blanco, G., Medical Biochemistry, 2017, Academic Press, London. [3] Gerich, J., Int. J. Gen. Med., 2013, 6, 877–895. [4] Krentz, A. J., Bailey, C. J., Drugs, 2005, 65 (3), 385-411. [5] Rosak, C., Mertes, G., Diabetes Metab. Syndr. Obes., 2012, 5, 357–367. [6] McCue, P. P., Shetty, K., Asia Pac. J. Clin. Nutr., 2004, 13 (1), 101–106. [7] Mohamed, E.A.H., Siddiqui, M.J.A., Ang, L.F., Sadikun, A., Chan, S.H., Tan, S.C.,

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the potential role of rosmarinic acid and sinensetin as α

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