Phenolic compounds of Heliotropium europaeum and their biological activities
Muneera S M. Al-Saleem1, Lamya H Al-Wahaibi1, Md Tabish Rehman2, Mohamed F AlAjmi2, Rawiyah A Alkahtani1, Wael M Abdel-Mageed3
1 Department of Chemistry, Science College, Princess Noura Bint Abdul Rahman University, Riyadh, Saudi Arabia
2 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
3 Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt, Saudi Arabia
|Date of Submission||21-Aug-2019|
|Date of Decision||12-Dec-2019|
|Date of Web Publication||31-Mar-2020|
Wael M Abdel-Mageed
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451; Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Heliotropium europaeum L. is known to contain pyrrolizidine alkaloids and possesses a wide variety of biological activities. Objective: The objective of the study was to discover antioxidant phytochemical compounds in H. europaeum and assess their in vitro lipoxygenase (LOX)- and angiotensin-converting enzyme (ACE) inhibitory activities. Materials and Methods: H. europaeum herbs were extracted exhaustively by maceration using 90% aqueous ethanol. Solvent fractionation with n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol was performed with the dried extract until complete exhaustion. Two antioxidant active fractions (CH2Cl2and EtOAc) were combined and subjected to medium pressure liquid chromatography followed by reversed-phase high-performance liquid chromatography. Quantitative evaluation in terms of the antioxidant activities of the isolated compounds was performed using the 2,2-diphenyl-1-picrylhydrazyl assay. The potential limiting effects on the action of ACE and soybean LOX were assessed quantitatively. Results: Isolates of eight newly identified phenolic compounds from H. europaeum exhibited antioxidant and ACE- and LOX-inhibitory activities. The isolated compounds were identified as kaempferol (1), luteolin (2), quercetin (3), kaempferol-3-O-glucoside (4), and luteolin-7-O-glucoside (5), in addition to caffeic acid (6), rosmarinic acid (7), and methyl rosmarinate (8). Among the isolated compounds, quercetin possessed the most potent antioxidant activity (IC50= 8.1 μM) and ACE-inhibitory activity (IC50= 17.5 μM), whereas rosmarinic acid and its methyl ester showed the strongest LOX-inhibitory activity (IC50s = 4.2 μM and 3.6 μM, respectively). Conclusion: Our study is the first report on the phenolic constituents of the H. europaeum herbal extract and their biological activities. A total of eight phenolics were identified in this extract and isolated for the first time. Our results on H. europaeum extract constituents provide some scientific evidence on the beneficial effects of its traditional uses.
Keywords: Angiotensin-converting enzyme inhibition, antioxidant, Boraginaceae, Heliotropium europaeum, lipoxygenase inhibition, rosmarinic acid
|How to cite this article:|
M. Al-Saleem MS, Al-Wahaibi LH, Rehman MT, AlAjmi MF, Alkahtani RA, Abdel-Mageed WM. Phenolic compounds of Heliotropium europaeum and their biological activities. Phcog Mag 2020;16, Suppl S1:108-16
|How to cite this URL:|
M. Al-Saleem MS, Al-Wahaibi LH, Rehman MT, AlAjmi MF, Alkahtani RA, Abdel-Mageed WM. Phenolic compounds of Heliotropium europaeum and their biological activities. Phcog Mag [serial online] 2020 [cited 2021 Jun 17];16, Suppl S1:108-16. Available from: http://www.phcog.com/text.asp?2020/16/68/108/281703
- A total of eight phenolic compounds were identified from the herbal extract of Heliotropium europaeum for the first time
- Quercetin possessed the most potent antioxidant and angiotensin-converting enzyme (ACE)-inhibitory activities
- Rosmarinic acid and its methyl ester showed the strongest lipoxygenase (LOX)-inhibitory activity. The flavonoid glycosides exhibited weak ACE- and LOX-inhibitory activities.
Abbreviations used: LOX: Lipoxygenase; ACE: Angiotensin-converting enzyme; MPLC: Medium pressure liquid chromatography; DPPH: 2,2-diphenyl-1-picrylhydrazyl; CVD: Cardiovascular disease; ROS: Reactive oxygen species; HRESIMS: High-resolution electrospray ionization mass spectrometry; NMR: Nuclear magnetic resonance; TLC: Thin layer chromatography; RP: Reversed phase; His: Histidine; Glu: Glutamic acid; Asp: Aspartic acid; Lys: Lysine; Tyr: Tyrosine; Val: Valine; Thr: Threonine; Ser: Serine; Phe: Phenylalanine; Ala: Alanine; SD: Standard deviations; Arg: Arginine; Gln: Glutamine; Trp: Tryptophan; SAR: Structural activity relationship.
| Introduction|| |
Cardiovascular disease (CVD) accounts for majority of the death cases reported in modern society. Hypertension, the most critical risk factor of CVD renal disorders, is called the silent killer affecting one-quarter of the adult population globally and one-third of people residing in the Western countries., Hypertension is still not adequately controlled and approximately 60% of the hypertension cases are either undetected or inadequately treated. Furthermore, drug therapy for hypertension management also relies on lifestyle modifications. The angiotensin-converting enzyme (ACE) inhibitors are first-line drugs in hypertension treatment.
Maintenance of the arterial blood pressure involves a crucial role of the renin–angiotensin–aldosterone system. Of the various components that play a central role in the system, ACE acts to convert angiotensin I into the active vasoconstrictor angiotensin II and also degrades bradykinin, a potent vasodilator. This helps to regulate the arterial blood pressure and electrolyte balance.,,, ACE inhibitors are typically used to control hypertension and to treat general cardiovascular and renal diseases, such as acute myocardial infarction, congestive heart failure, asymptomatic left ventricular dysfunction, and renal dysfunction.,,,
Many chemically synthesized ACE inhibitors, such as captopril and enalapril that helps to control hypertension, have several undesirable side effects, including angioneurotic edema, renal impairment, loss of taste, and cough., Thus, the search for natural ACE inhibitors is critical to discover safe and economical alternatives to synthetic drugs.
A large number of reactive oxygen species (ROS) are produced in the case of CVD diseases and hypertension. Studies have indicated an antithetical relationship prevails between antioxidant intake and mortality due to cardiovascular disorder.,, Therefore, expanding the search for natural therapeutics with antioxidants could identify drugs that render protection to the body from the free radicals and subsequently the development of many persistent cardiovascular disorders.,
The genus Heliotropium (Boraginaceae) comprises 250 species, which are found in warm regions. Heliotropium europaeum L., also known as the common heliotrope, European heliotrope, barooga weed, and caterpillar weed, is a herbaceous annual plant, native to Europe, Asia, North Africa, Australia, and Northern America.H. europaeum possesses a wide variety of biological activities, including antibacterial, antifungal, antitumor, anti-inflammatory, insecticidal, antispasmodic, cholagogue, emmenagogue, antipyretic, and anthelmintic effects and is used externally to treat warts and to promote wound healing.,,,,H. europaeum is known to contain pyrrolizidine alkaloids, such as heliotrine, heleurine, supinine, europine, and lasiocarpine, which are linked to the reported hepatotoxic, teratogenic, pulmotoxic, and mutagenic effects., Many reported cases of H. europaeum poisoning in sheep have resulted from ingesting food contaminated with seeds of this species.,, In India, Hong Kong, and South America, there have been some reports of human poisoning by H. europaeum due to the consumption of contaminated herbal teas with its seed hepatotoxic pyrrolizidine alkaloids.,,,,
Phytochemical analysis of H. europaeum indicated the presence of alkaloids, terpenoids, steroids, saponins, flavonoids, phenols, and tannins., Most of the previous reports were focused on the alkaloid content and the associated toxic effects, while other active metabolites and their beneficial biological activities were mostly neglected.
In continued search for drug leads from natural sources, this study extensively investigated dried H. europaeum herb to characterize its phenolic compounds. To our knowledge, this study is the first detailed analysis of the phenolic constituents of H. europaeum and their potential pharmacological activities. The aim of the current study is to evaluate the antioxidant and ACE- and lipoxygenase (LOX)-inhibitory actions of the phenolic metabolites obtained from the herbal extracts of H. europaeum.
| Materials and Methods|| |
Ultraviolet (UV) spectral analyses were carried out using Varian Cary ® 50 UV-Vis Spectrophotometer. High-resolution electrospray ionization mass spectrometry analysis was performed using a Bruker micrOTOF mass spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance DRX600 spectrometer (Bruker GmbH, Rheinstetten, Germany). Silica gel was utilized for performing column chromatography (Merck 60 Š, 70–230 mesh ASTM, Darmstadt, Germany). Purification was carried out using flash medium-pressure liquid chromatography (22 mm, i.d. × 30 cm, Kusano Scientific Co., Tokyo, Japan). Silica gel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) and RP-18 F254S plates (0.25 mm, Merck, Darmstadt, Germany) were used for thin-layer chromatography (TLC). Reversed-phase high-performance liquid chromatography (HPLC) was conducted using a Phenomenex Luna C18 column (Luna C18, 5 μm, 250 mm × 10 mm) on a Shimadzu HPLC-LC-20 AD binary gradient pump (Tokyo, Japan) combined with a SPD-M20A detector.
H. europaeum herbs were collected on March 2016 from the garden of medicinal plant, Pharmacognosy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The identity and authenticity of the plants were confirmed by an adept taxonomist at the Herbarium Unit and the voucher specimen (SY 017/2016) has been kept at the herbarium of the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
Extraction and isolation
Air-dried powdered plant material weighing 450 g was extracted three times by maceration using 2 L of 90% aqueous ethanol. Dry extract of 67 g (14.9%) was obtained from the alcoholic extract through filtration and concentration techniques performed under conditions of reduced pressure at 40°C. The dry material was suspended in 750 ml of distilled water and thereafter solvent fractionation was performed with n-hexane, followed by dichloromethane (CH2 Cl2), ethyl acetate (EtOAc), and finally, n-butanol until complete exhaustion. The process yielded the n-hexane fraction (HEPM1; 21.2 g), CH2 Cl2 fraction (HEPM2; 13.6 g), EtOAc fraction (HEPM3; 17.2 g), and n-butanol fraction (HEPM4; 6.1 g).
The CH2 Cl2 (HEPM2) and EtOAc (HEPM3) fractions were combined due to their similarities on TLC and were chromatographed using flash medium-pressure chromatography on a normal silica gel column and CHCl3–MeOH mixtures for separation according to the increasing polarity. TLC silica gel was used to keep a check on the 25 mL fractions that were collected using different solvent systems of CHCl3–MeOH (90:10) and CHCl3–MeOH–H2O (80:20:2) and (70:30:3), and 0.2% 2,2-diphenyl-1-picrylhydrazyl (DPPH) in MeOH and/or 15% v/v H2 SO4 in ethanol were used as spraying agents. Thereafter, the fractions were pooled to obtain nine main fractions (A–I) and were separated by reversed-phase HPLC on a Phenomenex Luna C18 column (250 mm × 10 mm, 5 μm) using a 5%–100% CH3 CN–H2O gradient over 40 min, yielding the following compounds: rosmarinic acid (7) and methyl rosmarinate (8), 16.1 mg and 7.9 mg yellow amorphous powders, respectively, from fraction C; kaempferol (1), luteolin (2), and quercetin (3), 10.2, 12.5, and 9.8 mg yellow amorphous powders, respectively, from fraction E; caffeic acid (6) 11.3 mg yellow amorphous powder from fraction G; and kaempferol-3-O-glucoside (4) and luteolin-7-O-glucoside (5), 12.7 and 8.5 mg yellow amorphous powders, respectively, from fraction H.
2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay
Rapid TLC screening method was used to qualitatively assess the radical-scavenging activities of the isolated compounds against 0.2% DPPH* (Sigma-Aldrich Chemical Co., St. Louis, USA) in MeOH. Quantitative determination with ascorbic acid as a positive control was carried out according to previously described methods.,
Angiotensin-converting enzyme inhibition assay
The ACE-inhibitory activity was determined spectrophotometrically using rabbit lung ACE (EC 188.8.131.52) (Sigma-Aldrich, Munich, Germany) and substrate (Hippuryl-histidyl-leucine [Bz-Gly-His-Leu]) (Sigma-Aldrich, Munich, Germany) adopting a previously described procedure of Cushman and Cheung method. All the experiments were performed thrice. ACE inhibitor captopril (Sigma-Aldrich, Munich, Germany) was used a positive control. Data were expressed as mean ± standard deviation; negative control reactions were performed without the test material and the resulting values were used to calculate the ACE-inhibitory activity expressed as percentage inhibition.
Percentage inhibition = ([Abscontrol − AbsSamples]/AbsControl) × 100
Lipoxygenase inhibition assay
The LOX-inhibitory activity of isolated phenolics was assessed spectrophotometrically using the soybean LOX (EC 184.108.40.206) type 1-B (Sigma-Aldrich, Munich, Germany) with linoleic acid (Sigma-Aldrich, Munich, Germany) as substrate adopting the method of Tappel. The reaction was performed using SpectraMax Plus 384 (Molecular Devices, USA). EZ-Fit Enzyme kinetics program was utilized to calculate the IC50 value (Perrella Scientific Inc., Amherst, USA). All the reactions were carried out in triplicate. The formulae used to calculate the percentage (%) of LOX inhibition was ([C − S]/C) × 100, where C and S are the LOX activity without and with the test compound, respectively.
AutoDock4.2 was used to perform molecular docking between ligands (Kaempferol, Luteolin, Quercetin, Caffeic acid, Rosmarinic acid, and Methyl rosmarinate) and protein (ACE) as described earlier., Briefly, the structures of ligands were downloaded from the PubChem database and were prepared for docking by adding Gasteiger partial charges, assigning rotatable bonds, and merging non-polar hydrogen atoms. The energy of each ligand was minimized using Universal Force Field with the help of Open Babel. The three-dimensional coordinate of ACE was retrieved from the RCSB database (Pdb Id: 1O8A, 2.0 Š resolution). The structure of ACE was optimized for docking by adding essential hydrogen atoms, Kollman united atom type charges, and solvation parameters. A grid of 63 × 73 × 67 Š dimensions with 0.375 Š spacing was created using AutoGrid. Molecular docking was performed using Lamarckian Genetic Algorithm and Solis and Wets local search methods. A total of 2,500,000 energy calculations were computed for each run and a total of ten runs were performed for each ligand. All the other parameters were set to default values. Further, the binding affinity (Kb) was computed from binding energy using the following relation.
Where R and T are the universal gas constant and temperature, respectively.
| Results|| |
The phytochemical examination of the herbal extract of H. europaeum guided by the results of the DPPH antioxidant assay facilitated separation and identification of eight phenolics (1–8), including five flavonoids, kaempferol (1), luteolin (2),, quercetin (3), kaempferol-3-O-glucoside (4), and luteolin-7-O-glucoside (5), in addition to caffeic acid (6), rosmarinic acid (7), and methyl rosmarinate (8) [Figure 1]. Light was shed on their structures through extensive NMR analyses, precise mass measurement, and by collating with the published data.
Quantitative assessment of the antioxidant activity of the isolated phenolics using the DPPH assay [Table 1] revealed that compounds kaempferol, luteolin, and quercetin (1-3) and luteolin-7-O-glucoside, caffeic acid, rosmarinic acid, and methyl rosmarinate (5–8) showed significant free radical scavenging activity. Quercetin (3) with an IC50 of 8.1 μM was identified to be more formidable than the positive control, ascorbic acid.
The results of the ACE-inhibitory activity test were expressed in percentage inhibition of ACE. Interestingly, all compounds possessed ACE-inhibitory activity in a concentration-dependent manner [Table 1]. Among all isolated compounds, quercetin (3) with an IC50 of 17.5 μM exhibited the highest ACE-inhibitory effect.
Furthermore, the anti-inflammatory activities of the phenolic isolates were assessed against the soybean LOX (EC 220.127.116.11), as summarized in [Table 1]. The findings revealed that both the free flavonoids (1–3) as well as the caffeic and rosmarinic acid derivatives (6–8) possess strong LOX-inhibitory activity with IC50 values within the range of 3.6–31.2 μM. The strongest activity was observed for rosmarinic acid (7) and its derivative methyl rosmarinate (8) with IC50 values of 4.2 μM and 3.6 μM, respectively.
In this study, molecular docking of the isolated phenolics kaempferol (1), luteolin (2), quercetin (3), caffeic acid (6), rosmarinic acid (7), and methyl rosmarinate (8) with ACE was performed to gain an insight into their mechanism of ACE inhibition. The data from docking experiments suggested that all the selected compounds were bound to the active site of ACE and hence acted as a potential inhibitor of ACE activity [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7] and [Table 2], [Table 3]. According to the molecular docking, kaempferol interacted with ACE by forming one Pi-Donor hydrogen bond with His383, four conventional hydrogen bonds (Glu376, Asp415, Lys454, and Tyr523), two Pi–Pi stacked hydrophobic interactions (His383 and Tyr523), one Pi–Pi T-shaped hydrophobic interaction (His383), and one Pi-alkyl hydrophobic interaction (Val379). Other amino acid residues such as Thr282, Ser284, His353, Asp453, His513, and Phe527 also interact with ACE and stabilize the ACE-kaempferol complex [Figure 2]. The kaempferol-ACE complex was stabilized by −8.6 kcal/mol of binding energy, which corresponded to a binding affinity of 2.03 × 106/M [Table 2]. Similarly, luteolin formed one carbon–hydrogen bond with Ser355, three conventional hydrogen bonds (Glu143, His513, and Tyr523), two Pi–Pi T-shaped hydrophobic interactions (His387 and Phe512), and one Pi-alkyl hydrophobic interaction (Val518) with ACE [Table 2]. Other amino acid residues involved in stabilizing the luteolin-ACE complex were Asn70, His353, Ala356, Lys368, His383, Phe391, Ser516, Val518, and Arg522 [Figure 3]. The binding energy and the corresponding binding affinity of luteolin and ACE complex were −8.6 kcal/mol and 2.03 × 106, respectively [Table 2]. Furthermore, quercetin formed four hydrogen bonds (Thr282, His353, and Lys454; Lys454 formed two hydrogen bonds), one Pi–Pi stacked hydrophobic interaction (Tyr523), one Pi–Pi T-shaped hydrophobic interaction (His383), and one Pi-alkyl hydrophobic interaction (Val379) with ACE. Other amino acid residues involved in stabilizing the quercetin-ACE complex were Gln281, Ser284, Glu376, Asp415, Asp453, His513, Tyr520, and Phe527 [Figure 4]. The binding energy and the corresponding binding affinity of quercetin and ACE complex were −9.0 kcal/mol and 3.99 × 106, respectively [Table 2].
|Figure 2: Molecular docking and interaction pattern of kaempferol with angiotensin-converting enzyme|
Click here to view
|Figure 3: Molecular docking and interaction pattern of luteolin with angiotensin-converting enzyme|
Click here to view
|Figure 4: Molecular docking and interaction pattern of quercetin with angiotensin-converting enzyme|
Click here to view
|Figure 5: Molecular docking and interaction pattern of caffeic acid with angiotensin-converting enzyme|
Click here to view
|Figure 6: Molecular docking and interaction pattern of rosmarinic acid with angiotensin-converting enzyme|
Click here to view
|Figure 7: Molecular docking and interaction pattern of methyl rosmarinate with angiotensin-converting enzyme|
Click here to view
|Table 2: Molecular docking parameters for the interaction between flavonoids (1-3) and angiotensin-converting enzyme|
Click here to view
|Table 3: Molecular docking parameters for the interaction between phenolic compounds (6-8) and angiotensin-converting enzyme|
Click here to view
Likewise, caffeic acid interacted with ACE by forming five hydrogen bonds (His353, Ala354, Asp415, His513, and Tyr523), one electrostatic interaction (Asp415), one Pi–Pi stacked hydrophobic interaction (His383), and one Pi-alkyl hydrophobic interaction (Val380). Other amino acid residues such as Val379, Glu384, Lys454, and Phe527 also interact with ACE and stabilize the ACE-caffeic acid complex [Figure 5]. The caffeic acid-ACE complex was stabilized by −6.5 kcal/mol of binding energy, which corresponded to a binding affinity of 5.85 × 104/M [Table 3]. Moreover, rosmarinic acid interacted with ACE by forming one Pi-donor hydrogen bond (His383), six conventional hydrogen bonds (Asn277, Gln281, Thr282, Glu376, and Glu384; Thr282 formed two hydrogen bonds), and one Pi-sigma hydrophobic interaction (Val380), one Pi–Pi stacked hydrophobic interaction (His353), four Pi–Pi T-shaped hydrophobic interaction (His383, Phe457, and Phe527; His383 formed two Pi-Pi T-shaped hydrophobic interactions), and one Pi-alkyl hydrophobic interaction (Ala354). Other amino acid residue stabilizing rosmarinic acid-ACE complex were Ser284, Val379, Asp415, Asp453, and Tyr523 [Figure 6]. The binding energy of rosmarinic acid for ACE was −8.5 kcal/mol which corresponded to a binding affinity of 1.72 × 106/M [Table 3]. Finally, methyl rosmarinate formed two hydrogen bonds (Gln281 and Tyr523), one electrostatic interactions (Zn 2+), one Pi–Pi stacked hydrophobic interaction (His383), and three Pi-alkyl hydrophobic interactions (Ala354 and Val380; Ala354 formed two Pi-alkyl hydrophobic interactions) with ACE. Other amino acid residues involved in stabilizing the quercetin-ACE complex were Glu162, Thr282, Cys352, His353, Gln369, Asp377, Val379, Glu384, Asp415, Lys454, His513, and Phe527 [Figure 7]. The binding energy and the corresponding binding affinity of methyl rosmarinate and ACE complex were −8.2 kcal/mol and 1.03 × 106, respectively [Table 3].
| Discussion|| |
In this study, eight phenolic compounds isolated for the first time were identified in the herbal extract of H. europaeum. They include five flavonoids in addition to caffeic acid (6) and rosmarinic acid as well as its methyl derivative (7 and 8). The first report that examines the main phenolic constituents in H. europaeum herbal extracts and assesses their biological activities are hereby presented.
The isolated phenolics possessed strong radical-scavenging activity. The antioxidant activity depended on the presence of the (ortho 3',4'-dihydroxy) (catechol) structure as found in compounds 2, 3, and 5–8. This could explain the weak scavenging activity of kaempferol-3-O-glucopyranoside (4) (IC50 >100) which lacks the catechol substructure in ring B.,, The (catechol) structure in the B-ring bestows a high stability to the flavonoid phenoxyl radicals through hydrogen bonding and/or via expanding electron delocalization., Furthermore, the presence of the α,β-unsaturated carbonyl moiety and α-hydroxyketone groups at ring C potentiated the antioxidant activity of flavonoids as observed with compound (3) through their chelating properties and enhancement of the radical stabilization via electron delocalization over all three-ring systems after the initial oxidation steps.,,, The glycosylation of the free hydroxyl group at C-3 (α-hydroxyketone) position reduces the antioxidant activity as observed with the compound (6).,,,
For ACE-inhibitory activity, the isolated phenolic compounds were tested and quercetin exhibited the most potent ACE inhibitors (IC5017.5 μM). In fact, the renin-angiotensin system acts as a crucial component in the process of controlling ion-water balance, blood pressure regulation, and pathophysiology of hypertension and renal failure. Renin acts through converting angiotensinogen to angiotensin I, which is converted subsequently to angiotensin II by ACE. The production of aldosterone from the adrenal cortex is triggered by the resulting angiotensin II. Aldosterone, in turn, raises the blood pressure via retention of sodium. Thus, ACE inhibition is considered an effective way of managing renal dysfunction and hypertension. Novel natural ACE inhibitors such as flavonoids or phenolic compounds are promising alternatives to the synthetic ACE inhibitors, such as enalapril or captopril, which cause many side effects during treatment of heart failure and essential hypertension in humans. In recent times, ACE has emerged as a critical target in the development of inhibitors/drugs to treat hypertension, heart failure, myocardial infarction, and diabetic nephropathy.
An insight into the mode of binding, and hence, inactivating ACE was gained by performing molecular docking of the isolated compounds with ACE. The active site of ACE comprises three parts: a carboxylate group crucial for enzymatic activity, a pocket that accommodates a hydrophobic side chain of C-terminal amino acid residues, and a zinc ion. Interaction between the zinc ion and the carbonyl of the penultimate peptide bond of the substrate (e.g., hippuryl-histidyl-leucine) results in its polarization and thereby poses for an electrophilic attack. The amino acid residues lining the active site of ACE comprises Tyr224, His353, Ala354, His383, Glu384, His387, Glu411, Lys511, His513, Tyr520, Arg522, and Tyr523. ACE has a characteristic HEXXH zinc-binding motif where His383 and His387 are directly coordinated with the zinc. In addition, Glu411 provides addition coordination to zinc through acetate ion from the solvent medium. The hydrolysis of substrate by ACE is initiated by two chloride ions; first, Cl - ion is bound to Arg186, Trp485, Arg489, and a water molecule, while the second Cl - ion is bound to Tyr224, Arg522, and a water molecule. Moreover, the X-ray crystal structure of ACE bound with a potential inhibitor (lisinopril) shows that several amino acid residues such as Glu162, Glu384, Lys511, Val518, and Tyr520 contribute significantly in the recognition and binding of the inhibitor.
ACE is a Zn-containing metalloenzyme, in which Zn ion at the active site is essential for ACE catalysis. Thus, the interaction of any ligand with Zn or the residues of ACE interacting with Zn ion is considered a significant parameter for the inhibition of ACE activity. The results of this study indicate that all the isolated compounds interacted with Zn ion or at least one amino acid residue of the HEXXH zinc-binding motif. Kaempferol interacted with the key residues of ACE such as His383 through electrostatic and hydrophobic interactions, His513 through hydrogen bonding, and Tyr523 via hydrophobic interaction. Likewise, luteolin interacted with some key residues of ACE through hydrogen bonding (His387, Glu411, and Tyr523) and hydrophobic interactions (His387 and Val518). Similarly, quercetin interacted with some key residues of ACE through hydrogen bonding (Tyr520 and Tyr523) and hydrophobic interactions (His383 and Tyr523). Caffeic acid was also bound to the active site of ACE and inhibited it by interacting with the key active site and catalytic residues such as His353, Ala354, Glu384, His513, and Tyr523 through hydrogen bonding, and His383 via electrostatic and hydrophobic interactions. Moreover, rosmarinic acid also interacted with key residues of ACE through hydrogen bonding (Ala354 and Glu384), carbon–hydrogen bonding (His383), and hydrophobic interactions (His353, Ala354 and His383). Further, methyl rosmarinate was also bound to the active site of ACE and inhibited it by binding with its key catalytic residues through hydrogen bonding (His353 and His383), carbon–hydrogen bonding (His513), and electrostatic (His383) and hydrophobic interactions (His383). The binding energy of all the isolated compounds was in the range of −6.5-9.0 kcal/mol, which corresponded to a binding affinity of 5.85 × 104- to 3.99 × 106/M. The results suggested that these compounds have a high probability to bind to the active site of ACE and thus act as a potential inhibitor of ACE activity. Moreover, the ACE-compound complex was stable in nature, thereby implying a low dissociation rate. The previous report by Al Shukor et al. also suggested that natural compounds such as gallic acid, protocatechuic acid, and caffeic acid act as an inhibitor of ACE by interacting with Zn ion through their carboxylate groups, while syringic acid interacts with Zn ion of ACE through its hydroxyl group. On the other hand, some natural compounds such as rutin, resveratrol, catechol, and pyrogallol inhibit ACE activity through an interaction with amino acid residues of the active site.
Many studies reported that phenolic compounds including flavonoids exert the ACE-inhibitory activity via the zinc chelate complex within the ACE active center.,,, The presence of an ortho-dihydroxy group, the catechol group in the flavonoids, is essential for zinc chelation and ACE-inhibitory activity. The reported structural activity relationship of flavonoids revealed that the 3',4'-catechol B-ring pattern was accompanied by the conjugated 4-oxo and α,β-unsaturated (2-3) bond, as found in luteolin (2) and quercetin (3) which are essential for the activity. Compounds (6–8) exhibit similarities with the flavonoids in terms of the presence of ortho-dihydroxy and α,β-unsaturated ester groups. The α,β-unsaturated esters are electrophiles and can bind to the enzyme with its carbonyl group that is coordinated to the zinc ion, and then, a Michael-type addition reaction may occur on the α,β-unsaturated acid or ester by the catalytically active carboxylate in ACE. The reaction results in irreversible inhibition of the enzyme.
The anti-inflammatory activity tests revealed that the free flavonoids (1–3) as well as the caffeic acid and rosmarinic acid with its derivative (6–8) were potent LOX inhibitors. The LOX-inhibitory effect was decreased by adding a sugar group to the core structure. The introduction of the sugar group increased the hydrophilicity, leading to a low affinity toward the lipophilic active site of LOX.,, These activities may contribute to the anti-inflammatory effects of H. europaeum and support its use in treating many inflammatory conditions.
It should be noted that methyl rosmarinate had slightly better antioxidant and anti-inflammatory activities than rosmarinic acid. This indicated that the methyl esterification might be favorable for improving both the activities. These findings are corroborated by many previous studies.,, Furthermore, several studies have reported that the esterification of rosmarinic acid suppresses its ionization at the physiological pH in vivo, which enhances its cellular uptake and intracellular biological activity. The present findings verified a good correlation of intrinsic antioxidant activity between cellular and non-cellular assays and suggested that further studies on methyl rosmarinate, rather than rosmarinic acid, may generate promising results.
| Conclusion|| |
Eight phenolic compounds were identified in the herbal extract of H. europaeum and isolated for the first time. They include five flavonoids in addition to caffeic acid, rosmarinic acid as well as its methyl derivative. The isolated phenolics possessed strong antioxidant, ACE-inhibitory, and anti-inflammatory activities. Quercetin possessed the most potent antioxidant and ACE-inhibitory activities, whereas rosmarinic acid and its methyl ester showed the strongest LOX-inhibitory activity.
For the ACE-inhibitory activity, the flavonoids and phenolic acids bind at the active site and interact with some key residues of ACE with high affinity. The ACE inhibitory activity of the isolated compounds was in the following order: 3 > 2 > 7 > 8 > 1 > 6. This order can be explained by understanding that the ortho-dihydroxy and α,β-unsaturated oxo groups that are essential for activity. Thus, these compounds hold the promise to be used directly or developed into more potent ACE inhibitors. Our results on H. europaeum extract constituents provided some scientific evidence on the beneficial effects of its traditional uses and could be considered as a promising drug candidate.
Financial support and sponsorship
This work was funded by the deanship of scientific research at Princess Nourah Bint Abdul Rahman University through the Research Groups Program Grant number (RGP-1440-0014).
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Heart Association. American Heart Association Heart Disease and Stroke Statistics: 2004 Update. Dallas, TX, American Heart Association; 2004.
Mittal BV, Singh AK. Hypertension in the developing world: Challenges and opportunities. Am J Kidney Dis 2010;55:590-8.
Kearney PM, Whelton M, Reynolds K, Muntner P, Whelton PK, He J. Global burden of hypertension: Analysis of worldwide data. Lancet 2005;365:217-23.
Mancia G, Bombelli M, Lanzarotti A, Grassi G, Cesana G, Zanchetti A, et al
. Systolic vs. diastolic blood pressure control in the hypertensive patients of the PAMELA population. Pressioni Arteriose Monitorate E Loro Associazioni. Arch Intern Med 2002;162:582-6.
Maruthur NM, Wang NY, Appel LJ. Lifestyle interventions reduce coronary heart disease risk: Results from the PREMIER Trial. Circulation 2009;119:2026-31.
Brown B, Hall AS. Renin-angiotensin system modulation: The weight of evidence. Am J Hypertens 2005;18:127S-33S.
Zhang R, Xu X, Chen T, Li L, Rao P. An assay for angiotensin-converting enzyme using capillary zone electrophoresis. Anal Biochem 2000;280:286-90.
Rosenthal J. Role of renal and extrarenal renin-angiotensin system in the mechanism of arterial hypertension and its sequelae. Steroids 1993;58:566-72.
Imig JD. ACE Inhibition and Bradykinin-Mediated Renal Vascular Responses: EDHF Involvement. Hypertension 2004;43:533-5.
Carson PE. Rationale for the use of combination angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker therapy in heart failure. Am Heart J 2000;140:361-6.
Borghi C, Cosentino E, De Sanctis D. Angiotensin-converting enzyme inhibition and cardiovascular prevention: More than twenty years of clinical success. Ital Heart J Suppl 2005;6:769-79.
Shlipak MG, Browner WS, Noguchi H, Massie B, Frances CD, McClellan M. Comparison of the effects of angiotensin converting-enzyme inhibitors and beta blockers on survival in elderly patients with reduced left ventricular function after myocardial infarction. Am J Med 2001;110:425-33.
Miller MA, Texter M, Gmerek A, Robbins J, Shurzinske L, Canter D. Quinapril hydrochloride effects on renal function in patients with renal dysfunction and hypertension: A drug-withdrawal study. Cardiovasc Drugs Ther 1994;8:271-5.
Israili ZH, Hall WD. Cough and angioneurotic edema associated with angiotensin-converting enzyme inhibitor therapy. A review of the literature and pathophysiology. Ann Intern Med 1992;117:234-42.
Antonios TF, MacGregor GA. Angiotensin converting enzyme inhibitors in hypertension: Potential problems. J Hypertens Suppl 1995;13:S11-6.
Aune D, Keum N, Giovannucci E, Fadnes LT, Boffetta P, Greenwood DC, et al
. Dietary intake and blood concentrations of antioxidants and the risk of cardiovascular disease, total cancer, and all-cause mortality: A systematic review and dose-response meta-analysis of prospective studies. Am J Clin Nutr 2018;108:1069-91.
Zhao LG, Shu XO, Li HL, Zhang W, Gao J, Sun JW, et al
. Dietary antioxidant vitamins intake and mortality: A report from two cohort studies of Chinese adults in Shanghai. J Epidemiol 2017;27:89-97.
Kim K, Vance TM, Chen MH, Chun OK. Dietary total antioxidant capacity is inversely associated with all-cause and cardiovascular disease death of US adults. Eur J Nutr 2018;57:2469-76.
Aruoma OI, Akanmu D, Cecchini R, Halliwell B. Evaluation of the ability of the angiotensin-converting enzyme inhibitor captopril to scavenge reactive oxygen species. Chem Biol Interact 1991;77:303-14.
Tundis R, Nadjafi F, Menichini F. Angiotensin-converting enzyme inhibitory activity and antioxidant properties of Nepeta crassifolia
Boiss & Buhse and Nepeta binaludensis
Jamzad. Phytother Res 2013;27:572-80.
Saeedi M, Morteza-Semnani K. Chemical composition and antimicrobial activity of the essential oil of Heliotropium europaeum
. Chem Nat Compd 2009;45:98-9.
Zargari A. Medicinal Plants. Vol. 3. Tehran, Iran: Tehran University Publications; 1992. p. 526-8.
Mahmood A, Qureshi RA, Mahmood A, Sangi Y, Shaheen H, Ahmad I, et al
. Ethnobotanical survey of common medicinal plants used by people of district Mirpur AJK Pakistan. J Med Plants Res 2011;5:4493-8.
Bonet MA, Vallès J. Ethnobotany of Montseny biosphere reserve (Catalonia, Iberian Peninsula): Plants used in veterinary medicine. J Ethnopharmacol 2007;110:130-47.
Qureshi R, Bhatti R, Memon R. Ethnomedical uses of herbs from northern part of Nora desert Pakistan. Pak J Bot 2010;42:839-51.
Duke JA, Bogenschutz-Godwin MJ, De Cellier J, Duke PA. Handbook of Medicinal Herbs. 2nd
ed. Florida, USA: CRC Press; 2002. p. 372.
Al-Snafi AE. Pharmacological and toxicological effects of Heliotropium undulatum
] and Heliotropium europaeum
– A review. IAJPS 2018;5:2150-8.
Shimshoni JA, Mulder PP, Bouznach A, Edery N, Pasval I, Barel S, et al
. Heliotropium europaeum
poisoning in cattle and analysis of its pyrrolizidine alkaloid profile. J Agric Food Chem 2015;63:1664-72.
Pass DA, Hogg GG, Russell RG, Edgar JA, Tence IM, Rikard-Bell L. Poisoning of chickens and ducks by pyrrolizidine alkaloids of Heliotropium europaeum
. Aust Vet J 1979;55:284-8.
Fu PP, Xia Q, He X, Barel S, Edery N, Beland FA, et al
. Detection of pyrrolizidine alkaloid DNA adducts in livers of cattle poisoned with Heliotropium europaeum
. Chem Res Toxicol 2017;30:851-8.
Diaz GJ. Toxicosis by Plant Alkaloids in Humans and Animals in Colombia. Toxins (Basel) 2015;7:5408-16.
Elvin-Lewis M. Safety issues associated with herbal ingredients. Adv Food Nutr Res 2005;50:219-313.
Wiedenfeld H, Edgar J. Toxicity of pyrrolizidine alkaloids to humans and ruminants. Phytochem Rev 2011;10:137-51.
Abdel-Mageed WM, Bayoumi SA, Radwan AA, Salem-Bekhit MM, Abd-Alrahman SH, Basudan OA, et al
. Simmondsia chinensis
: A rich source of bioactive flavonoids and lignans. Ind Crops Prod 2014;60:99-103.
Abdel-Mageed WM, Al-Wahaibi LH, Backheet EY, El-Gamal AA, Gouda YG, Basudan OA, et al
. New phenolic glycosides with cyclooxygenase inhibition from the roots of Tecoma mollis
. Phytochem Lett 2017;21:98-103.
Cushman DW, Cheung HS. Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem Pharmacol 1971;20:1637-48.
Tappel, AL. Methods in Enzymology. Vol. 5. New York: Academic Press; 1962. p. 539-42.
Rehman MT, Shamsi H, Khan AU. Insight into the binding mechanism of imipenem to human serum albumin by spectroscopic and computational approaches. Mol Pharm 2014;11:1785-97.
Hussain A, AlAjmi MF, Rehman MT, Khan AA, Shaikh PA, Khan RA. Evaluation of transition metal complexes of benzimidazole-derived scaffold as promising anticancer chemotherapeutics. Molecules 2018;23. pii: E1232.
Natesh R, Schwager SL, Sturrock ED, Acharya KR. Crystal structure of the human angiotensin-converting enzyme-lisinopril complex. Nature 2003;421:551-4.
Alajmi MF, Alam P, Rehman MT, Husain FM, Khan AA, Siddiqui NA, et al
. Interspecies anticancer and antimicrobial activities of Genus Solanum
and estimation of rutin by Validated UPLC-PDA Method. Evid Based Complement Alternat Med 2018;2018:6040815.
Scotti L, Fernandes MB, Muramatsu E, Emereciano VP, Tavares JF, Silva MS, et al
. 13C NMR spectral data and molecular descriptors to predict the antioxidant activity of flavonoids. Braz J Pharm Sci 2011;47:241-9.
Liu H, Mou Y, Zhao J, Wang J, Zhou L, Wang M, et al
. Flavonoids from Halostachys caspica
and their antimicrobial and antioxidant activities. Molecules 2010;15:7933-45.
Lu Y, Foo LY. The polyphenol constituents of grape pomace. Food Chem 1999;65:1-8.
Chiruvella KK, Mohammed A, Dampuri G, Ghanta RG, Raghavan SC. Phytochemical and antimicrobial studies of methyl angolensate and luteolin-7-o-glucoside isolated from callus cultures of Soymida febrifuga
. Int J Biomed Sci 2007;3:269-78.
Chang SW, Kim KH, Lee IK, Choi SU, Ryu SY, Lee KR. Phytochemical Constituents of Bistorta manshuriensis
. Nat Prod Sci 2009;15:234-24.
Abedini A, Roumy V, Mahieux S, Biabiany M, Standaert-Vitse A, Rivière C, et al
. Rosmarinic acid and its methyl ester as antimicrobial components of the hydromethanolic extract of Hyptis atrorubens
Poit. (Lamiaceae). Evid Based Complement Alternat Med 2013;2013:604536.
Sarian MN, Ahmed QU, Mat So'ad SZ, Alhassan AM, Murugesu S, Perumal V, et al
. Antioxidant and antidiabetic effects of flavonoids: A structure-activity relationship based study. Biomed Res Int 2017;2017:8386065.
Amić D, Davidović-Amić D, Beslo D, Rastija V, Lucić B, Trinajstić N. SAR and QSAR of the antioxidant activity of flavonoids. Curr Med Chem 2007;14:827-45.
Bors W, Heller W, Michel C, Saran M. Flavonoids as antioxidants: Determination of radical-scavenging efficiencies. Methods Enzymol 1990;186:343-55.
Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. J Nutr Biochem 2002;13:572-84.
Liu X, Fernandez M, Wouters MA, Heyberger S, Husain A. Arg (1098) is critical for the chloride dependence of human angiotensin I-converting enzyme C-domain catalytic activity. J Biol Chem 2001;276:33518-25.
Al Shukor N, Van Camp J, Gonzales GB, Staljanssens D, Struijs K, Zotti MJ, et al
. Angiotensin-converting enzyme inhibitory effects by plant phenolic compounds: A study of structure activity relationships. J Agric Food Chem 2013;61:11832-9.
Kwon EK, Lee DY, Lee H, Kim DO, Baek NI, Kim YE, et al
. Flavonoids from the buds of Rosa damascena
inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme a reductase and angiotensin I-converting enzyme. J Agric Food Chem 2010;58:882-6.
Loizzo MR, Said A, Tundis R, Rashed K, Statti GA, Hufner A, et al
. Inhibition of angiotensin converting enzyme (ACE) by flavonoids isolated from Ailanthus excelsa
(Roxb) (Simaroubaceae). Phytother Res 2007;21:32-6.
Oh H, Kang DG, Kwon JW, Kwon TO, Lee SY, Lee DB, et al
. Isolation of angiotensin converting enzyme (ACE) inhibitory flavonoids from Sedum sarmentosum
. Biol Pharm Bull 2004;27:2035-7.
Balasuriya BW, Rupasinghe HP. Plant flavonoids as angiotensin converting enzyme inhibitors in regulation of hypertension. Funct food health dis 2011;1:172-88.
Guerrero L, Castillo J, Quiñones M, Garcia-Vallvé S, Arola L, Pujadas G, et al
. Inhibition of angiotensin-converting enzyme activity by flavonoids: Structure-activity relationship studies. PLoS One 2012;7:e49493.
Sadik CD, Sies H, Schewe T. Inhibition of 15-lipoxygenases by flavonoids: Structure-activity relations and mode of action. Biochem Pharmacol 2003;65:773-81.
Li QL, Li BG, Zhang Y, Gao XP, Li CQ, Zhang GL. Three angiotensin-converting enzyme inhibitors from Rabdosia coetsa
. Phytomedicine 2008;15:386-8.
Adomako-Bonsu AG, Chan SL, Pratten M, Fry JR. Antioxidant activity of rosmarinic acid and its principal metabolites in chemical and cellular systems: Importance of physico-chemical characteristics. Toxicol In vitro
Bayrasy C, Chabi B, Laguerre M, Lecomte J, Jublanc E, Villeneuve P, et al
. Boosting antioxidants by lipophilization: A strategy to increase cell uptake and target mitochondria. Pharm Res 2013;30:1979-89.
Iqbal K, Nawaz SA, Malik A, Riaz N, Mukhtar N, Mohammad P, et al
. Isolation and lipoxygenase-inhibition studies of phenolic constituents from Ehretia obtusifolia
. Chem Biodivers 2005;2:104-11.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]