Isolation, Identification and Molecular Docking Studies of a New Isolated Compound, from Onopordon acanthium: A Novel Angiotensin Converting Enzyme (ACE) inhibitor

https://doi.org/10.1016/j.jep.2013.05.046Get rights and content

Abstract

Ethnopharmacological relevance

Onopordon acanthium (also known as Scotch thistle) is a medicinal plant of the Asteraceae family that is widely distributed in Europe and Asia. This plant has been long used in traditional medicine as a hypotensive, cardiotonic and diuretic agent.

Aim of the study

The present study is designed to isolate an active compound with ACE inhibition activity from O. acanthium, measure antioxidant activity, predict domain specificity and pharmacokinetic properties of the isolated compound.

Materials and methods

Methanolic extract of O. acanthium seeds, has been subjected to a repeated column chromatography to give a pure compound with Angiotensin Converting Enzyme (ACE) inhibition activity. The ACE inhibition activity was determined using hippuryl-L-histidyl-L-leucine (HHL) as substrate in an in vitro ACE assay. Structure of the pure compound, isolated from O. acanthium has been established by spectroscopic methods, including Infrared (IR), Nuclear Magnetic Resonance (NMR) and Mass spectrum analysis. In addition, antioxidant activity of the new isolated compound, was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and compared with those of BHT and Trolox as positive controls. Enzyme type inhibition and ACE-C or N domain specificity of the new compound was further evaluated through molecular modeling and docking studies.

Results

Structure of the pure compound, isolated from O. acanthium (83±1% ACE inhibition activity at concentration of 330 μg/ml), has been established. The isolated compound possessed acceptable antioxidant activity (IC50 value of 2.6±0.04 μg/ml) in comparison with BHT (IC50 value of 10.3±0.15 μg/ml) and Trolox (IC50 value of 3.2±0.06 μg/ml). Molecular docking predicted competitive type enzyme inhibition and approximately similar affinity of the isolated compound for ACE-C and N domains.

Conclusion

The results derived from computational and in vitro experiments, confirm the potential of the isolated compound, from O. acanthium as a new antihypertensive compound and give additional scientific support to an anecdotal use of O. acanthium in traditional medicine to treat cardiovascular disease such as hypertension.

Introduction

Hypertension is one of the greatest public health problems worldwide. It is predicted that by 2025, 60% of the world population will suffer hypertension (Kearney et al., 2005). Since hypertension, results in an increasing risk of developing cardiovascular diseases, kidney diseases (Johnson et al., 2007) and stroke (Stokes et al., 1987), developing new therapeutic approaches is critical (Carretero and Oparil, 2000). It has been accepted that angiotensin converting enzyme (ACE) plays an important role in blood pressure management (Skeggs et al., 1956). ACE (EC 3.4.15.1), a zinc metallopeptidase, cleaves the dipeptide His-Leu residue from the C-terminal of inactive decapeptide angiotensin I (Ang I) and converts it to the potent vasoconstrictor angiotensin II (Ang II). ACE also affects kallikrein–kinin system, by promoting the degradation of bradykinin, which is a potent vasodilator peptide involve in blood pressure management and inflammation (Campbell, 2003). ACE is expressed in two forms, somatic and germinal. Somatic ACE is composed of two catalytic domains (N- and C- domains) with 60% identity in sequence. Each domain consists of a functional active site and distinct functional properties. Germinal ACE exhibits only one domain which is identical to the C-domain of somatic ACE (Kim et al., 2003). Despite the structural identity of the two domains, there are some variations between the active sites, that results in a specific affinity for substrates and designing precise inhibitors. Lack of adequate C-domain specificity for ACE inhibitors, leads to bradykinin accumulation (Van Esch et al., 2005), which result in several adverse side effects associated, such as dry cough and angioedema (Morimoto et al., 2004). On the other hand, in hypotensive patients nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation is induced by Ang II that ends up in oxidative stress, an important factor in cardiovascular diseases and hypertension. So there is a superiority for an antihypertensive drug to possess antioxidant activity.

Medicinal plants are potential sources to discover new drugs. Therefore, searching for natural and selective ACE inhibitors as alternatives to synthetic ones, is of great interest. In our recent report (submit for publication), we have used an in vitro ACE assay to identify plants with ACE inhibition activity. We have found six medicinal plants with promising activities against ACE, including Scotch thistle (Onopordon acanthium L. family Asteraceae) with high antioxidant activity in comparison with butylated hydroxytoluene (BHT) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as positive controls. O. acanthium mostly grows in Europe, western and central Asia. The plant is up to 1.5 m tall, with hairy leaves, purple flowers and annual life cycle. O. acanthium also grows in Iran, mostly in Azarbayejan province (its local name is khaje bashi). The plant petals are used as a yellow food coloring and flavoring agent. The seeds are a good source for high quality edible oil, the stems are used as a vegetable, and the receptacle is eaten like an artichoke. O. acanthium is used in traditional medicine as an antibacterial, hemostatic, and hypotensive agent (Maugini, 1994, Mariani et al., 2009).

In this study, the active compound with ACE inhibition activity was isolated from O. acanthium, ACE-C and N domain affinity was predicted by molecular docking studies, and the antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.

Section snippets

Chemical reagents

Angiotensin converting enzyme (ACE) from rabbit lung, hippuryl-L-histidyl-L-leucine (HHL) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were purchased from Sigma-Aldrich Co. (England). 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2-aminoethyl diphenyl borate (natural product reagent), hydrochloric acid, potassium hydrogen phosphate, sodium chloride, dimethyl solfoxide (DMSO),

Identification of the new isolated compound from O. acanthium

Compound 1 (Fig. 1) was isolated from O. acanthium extract as a yellow crystal structure. This compound has been identified by IR, 1H NMR, 13C NMR and MASS analysis.

Compound 1: C18H14O6; mp 136–139 °C; IR (KBr) νmax cm−1 3463, 3409, 3318, 2928, 1647, 1623, 1575, 1531, 1245, 1200, 975 and 812 cm−1; 1H NMR (500 MHz, DMSO-d6): 2.77 (2 H, t, J=7.6 Hz, H4- isochromen), 3.41 (2 H, m, H3- isochromen), 6.33 (1 H, d, J=15.7 Hz, Hα-vinyl), 6.58 (1 H, dd, J=8.5 & 2.3 Hz, H6- phenyl), 6.74 (1 H, d, J=8.0 Hz, H5-

Discussion

It is accepted that using ACE inhibitors have some benefits in the treatment of heart failure, hypertension, and myocardial infarction. These days most of ACE inhibitors on the market have some adverse side effects. Specific ACE inhibition activity for C-domain, has resulted in developing new ACE inhibitors with fewer side effects. On the other hand, production of reactive oxygen species (ROS) that leads to oxidative stress, plays an important role in cardiovascular disease. Therefore,

Acknowledgements

The authors would like to acknowledge Ms. K. Bagherzadeh for kindly revising the manuscript. The financial supports of the Research Council of the Tehran University of Medical Sciences, is gratefully acknowledged.

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