Coumarins Isolated from Murraya paniculata in Vietnam and Their Inhibitory Effects against Enzyme Soluble Epoxide Hydrolase (sEH)

Abstract

In the search for bioactive constituents from Vietnam medicinal plants, the leaves and stems of Murraya paniculata collected in HoaBinh Province, Vietnam were selected for chemical investigation. From the n-hexane fraction, two sterols, including β-sitosterol (6) and stigmasterol (7), and from the chloroform fraction, five coumarins, including mexoticin (1), omphalocarpin (2), murrangatin (3), kimcuongin (4), and murracarpin (5), were obtained. The structures of the isolated compounds were determined from ESI-MS, HR-ESI-MS, and NMR (1D and 2D) spectroscopic data. Coumarins (1–5) were elucidated for inhibitory effects against soluble epoxide hydrolase. Among them, coumarins (2–4) showed soluble epoxide hydrolase inhibitory activity with IC₅₀ values 2.2 ± 4.7, 13.9 ± 6.5, and 3.2 ± 4.5 µM, respectively. A kinetic study of the five coumarins revealed the noncompetitive enzymatic mode for 3 and 4, and a mixture of competitive/noncompetitive enzymatic modes for coumarin 2. Using molecular modelling, the coumarin kimcuongin (4) showed the best binding outline into active sites of human soluble epoxide hydrolase.

Key words

Murraya paniculata - Rutaceae - coumarin - kimcuongin - murracarpin - AutoDock/Vina - soluble epoxide hydrolase (sEH)

Abbreviations

AUDA: 12-(3-adamantan-1-yl-ureido) dodecanoic acid

DHETs: dihydroxyeicosatrienoicdiols

EETs: epoxyeicosatrienoic acids

Ki: inhibitor constant

Km: Michaelis-Menten constant

PHOME: 3-phenyl-cyano(6-methoxy-2-naphthalenyl)methyl ester-2-oxiraneacetic acid

PLIP: protein-ligand interaction profiler

sEH: soluble epoxide hydrolase

The sEH is the main enzyme that catalyzes the metabolism of EETs into the more polar and usually less potent metabolites DHETs [1]. Early studies have indicated that chemical compounds that can inhibit the sEHsʼ activity and stabilize the endogenous EET levels may represent potential therapeutic agents for cardiovascular disease [2], [3] and the onset of several other diseases [4], [5].

Murraya paniculata (L.) Jack (Rutaceae), local name “Nguyet que”, is mostly grown as ornaments for its glossy green foliage and white fragrant flowers. It is used as a medicinal plant in Vietnamese traditional medicine. There were several publications of phytochemical studies of Murraya (see Supporting Information). In this study, we describe the inhibitory activity against the sEH of coumarins isolated from this plant and explain this effect by molecular modelling.

The methanol extract of the dry leaves of M. paniculata was suspended with water and subsequently fractioned with n-hexane, chloroform, and ethyl acetate. From the n-hexane fraction, two sterols, β-sitosterol (6) and β-stigmasterol (7), were isolated. From the chloroform fraction of the leaves of M. paniculata, five coumarins (1–5) were subsequently isolated through column chromatography including mexoticin (1) [6], omphalocarpin (2) [7], murrangatin (3) [8], kimcuongin (4), and (−)-murracarpin (5; [Fig. 1]). The latter two coumarins are reported to possess vasorelaxing activity [9].

Fig. 1 Structures of the isolated compounds (1–5).

The in vitro the inhibitory activity of the isolated coumarins (1–5) from M. paniculata leaves was investigated based on the hydrolysis of the sEH on an artificial fluorescent substrate, PHOME, with AUDA as a positive control (IC₅₀ 15.7 ± 2.7 nM; [Fig. 2]). With the exception of compounds 1 and 5, coumarins 2, 3, and 4 exhibited potential sEH inhibitory activity (assessed at the sEH concentration of 25 µM; [Table 1]). Their IC₅₀ values were found to be 3.2 ± 4.5, 13.9 ± 6.5, and 2.2 ± 4.7 µM, respectively ([Fig. 2] and [Table 1]).

Fig. 2 Inhibitory effect of the isolated coumarins (1–5) on sEH activity determined using the fluorometric method. The sEH activity is expressed as the percentage of control activity (n = 3). AUDA (IC₅₀ 15.7 ± 2.7 nM) was used as a positive control.

Table 1 A In vitro sEH inhibitory activity with IC₅₀ values, kinetic study of coumarins (1–5) and ν _max, K_m, and K_i,exp values [Substrate concentration of 0.75 µM. AUDA was used as a positive control 15.7 ± 2.7 (nM)]. B Predicted and experimental binding parameters of coumarins 2–4 to sEH enzyme.

No.	Compounds	A. Kinetic parameters					B. Predicted and experimental binding parameters
		IC50 (µM)	Binding mode	ν max	Km	Ki,exp =  1/Km	ΔG (kcal/mol)	Ki,pred	pKi, pred	IC50, pred (µM)	pKi,exp
ND = not determined
1	Mexoticin	> 25	ND
2	Omphalocarpin	2.2 ± 4.7	Mixture (competitive/noncompetitive)	520.6	4.922	0.203 169	− 6.4	2.02E-05 M	4.69	2.5	5.66
3	Murrangatin	13.9 ± 6.5	noncompetitive	628.8	4.903	0.203 956	− 6.8	1.03E-05 M	4.99	(ND)	4.87
4	Kimcuongin	3.2 ± 4.5	noncompetitive	651.8	3.573	0.279 876	− 7.8	1.89E-06 M	5.72	(ND)	5.49
5	Murracarpin	> 25	ND

Kinetic parameters including maximum velocity (ν _max), K_m, K_i, and the mode of inhibition of the potent sEH inhibitors 2, 3, and 4 were obtained using the sEH enzyme as shown in [Table 1]. According to the K_i,exp value, omphalocarpin (2) showed a higher possibility to interact with the sEH enzyme. In the presence of the two inhibitors 3 and 4, the K_m values of the sEH enzyme were found to be similar, while the ν _max and K_m values were gradually decreased and even lower than those of the reaction without the presence of the inhibitor (0.0 µM; [Fig. 3]). This suggested that the inhibitory activity of 3 and 4 on the sEH followed the noncompetitive binding mode and the coumarins might affect the enzyme-substrate complex. While in the case of the reaction catalyzed by coumarin 2, the ν _max value decreased, but the K_m value increased compared to that of the reaction without inhibitor. These facts suggest that the inhibitory activity of coumarin 2 on sEH was different from those in the presence of coumarins 3 and 4. It might follow a mixture (competitive/noncompetitive) binding mode.

Fig. 3 Effects of coumarins 2, 3, and 4 on the activity of enzyme sEH for hydrolysis of the PHOME substrate.

In order to investigate the binding mechanisms of the active coumarins to the sEH enzyme, molecular docking using AutoDock Vina software was carried out, where each coumarin was manually docked at the active site of the human sEH (complexed with ligands; [Fig. 4] and Tables 1 S and 2 S, Supporting Information). All three coumarins have hydrophobic interactions to acid amines Asp335A, Leu408A, Leu499A, His524A, and Trp525A ([Fig. 5]). The smallest hydrophobic bond belonged to omphalocarpin (2) binding to Leu499A with 2.66 Å. Kimcuongin (4) prefered to binding to phenylalanine [Phe267A (3.89 Å) and Phe381A (3.55 and 3.97 Å]. The estimated hydrogen bond formed between the carbonyl groups of the coumarins and the residues showed that the best docked conformation was determined for kimcuongin (4) with Tyr383A and Tyr466A of the sEH active site from 2.13–2.77 Å (Table 2 S, Supporting Information).

Fig. 4 A Docked complexes of omphalocarpin (2, blue), murrangatin (3, magenta), and kimcuongin (4, green) into human sEH shown in cartoon representation. B PLIP 3D-interaction map of the ligands with sEH residues playing an essential role for binding.

Fig. 5 ABS-Scan energy plot. ΔΔG values recorded after alanine scanning mutation of the single residues involved in the binding of omphalocarpin (blue), murrangatin (magenta), and kimcuongin (green).

During the docking process, the protein was considered to be rigid while the ligands and those amino acids inside the pocket were flexible. The AutoDock output results represented the docking scores as Gibbs free energy of binding (ΔG) values, further converted to the predicted inhibition constants (pK_i,pred). The designed compounds (2, 3, and 4) were found to have excellent binding affinity to the enzyme, showing binding energies as ΔG values of − 6.4, − 6.6 and − 7.8 kcal/mol, respectively. The negative values of ΔG indicated that the coumarins bind to sEH spontanously and these values also proved that the compounds possess potential sEH enzyme inhibitory binding activities. Three compounds occupy the same cavity (Fig. 1 S, Supporting Information) with only some differences in amino acid residue involvement because of their dihedral rotation and conformational mismatch. The small variance in ΔG values and binding posed inside the pocket may be attributed to the differences in the position of the functional groups in the selected compounds. The ΔG values were further converted to pK_i,pred, where K_i was calculated by the formula K_i = IC₅₀/1 + [S]/K_m = exp(ΔG/R × T), so IC₅₀ = exp(ΔG/R × T) × 1 + [S]/K_m ([Table 1]).

The docking results of the three compounds were analyzed by a bioinformatic tool, giving an interaction diagram and a table of interaction data for each binding site. For all compounds, the binding was dominated by hydrophobic interactions and hydrogen bonds. The three diagrams, in agreement with the ΔG calculation results, showed that compounds 2 and 3 had a similar set of binding profiles (seven hydrogen interactions and one hydrogen bond), while 4 had the best binding outline (nine hydrogen bonds and two hydrogen bonds; [Fig. 4]).

The amino acids presented at the binding site are computationally mutated to alanine in an ABS-Scan tool to perform an in silico alanine scanning mutagenesis for binding site residues in the protein-ligand complex ([Fig. 5]). The binding energy is computed for each mutant and the corresponding energy differencies (ΔΔG) values between the wild-type protein and the mutated one are also calculated. The ΔΔG profile shared by murrangatine (3) and kimcuongin (4) was not totally repeated by omphalocarpin (2), which showed the smallest binding energy. The docked poses of coumarin derivatives clearly demonstrated the binding positions of the ligand with the enzyme. The main binding force is due to the interactions of Tyr466 and Tyr383 with the coumarin derivatives, while other important interactions were found with amino acid residues Trp 525, Trp 336, and Phe 267. The coumarin kimcuongin (4), furthermore, showed good affinity towards Tyr 383, Gln 384, and Phe 387, which were responsible for better sEH binding energy and its potential inhibitory activity.

In summary, among five coumarins isolated from the chloroform fraction of the leaves and stems of M. paniculata, three coumarins, omphalocarpin (2), murrangatin (3), and kimcuongin (4), showed sEH inhibitory activity with IC₅₀ values 2.2 ± 4.7, 13.9 ± 6.5, and 3.2 ± 4.5 µM, respectively, in noncompetitive (3, 4) and mixture-kinetic mode (2). Using a computational approach, kimcuongin (4) showed the best binding outline, characterized by the smallest binding energy (ΔG = − 7.8 kcal/mol), a good affinity towards Tyr 383, Gln 384, and Phe 387, and its potential sEH inhibitory activity. These three coumarins might be worthy to further investigate in order to develop a new scaffold of therapeutic agents for cardiovascular diseases.

Materials and Methods

The leaves and twigs of M. paniculata were collected in Cuc Phuong National Park, Hoa Binh Province, North Vietnam. The plant was identified by botanist Dr. Tran The Bach, Institute of Ecology and Biological Resources (VAST). A voucher specimen (C-425) is deposited in the herbarium of the Institute of Natural Products Chemistry (VAST), Hanoi, Vietnam.

Dried powdered leaves and twigs of M. paniculata (3.2 kg) were extracted with MeOH to yield a black crude MeOH extract (120 g). The crude MeOH extract was suspended in hot MeOH-water (1 : 1, v/v) and successively partitioned with n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), and water to give the corresponding solvent-soluble fractions n-hexane (6.7 g), chloroform (7.2 g), EtOAc (16.7 g), and water (60 mL). Repeated chromatography of n-hexane and chloroform fractions on silica gel columns with different eluting solvents resulted in the separation of compounds 1–5 and 6, 7, respectively.

Supporting information

General experimental procedures, extraction and isolation, sEH bioassays, and molecular modelling are described in detail in Supporting Information.

Acknowledgements

This research was supported by the Ministry of Science and Technology (MOST) for the Vietnam-Korea Project (Code 52/2011/NĐT) and the National Foundation for Science and Technology Development of Vietnam (NAFOSTED), grant No. 104.01–2010.25.

Conflict of Interest

The authors declare no conflict of interest.

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