Soluble epoxide hydrolase (sEH, EC 3.3.2.10) is the major enzyme responsible for the hydrolysis of epoxy fatty acids (EpFAs) to their corresponding vicinal diols in humans and other mammals.1 These EpFAs include the epoxides of linoleic, arachidonic, eicosapentaenoic, and docosahexaenoic acid that are produced primarily by cytochrome P450s. These natural molecules are pleiotropic endogenous mediators with key functions in inflammation,1 pain,2 and blood pressure regulation.3 Increasing the levels of endogenous EpFAs by inhibiting sEH has been shown to block and resolve inflammation,4 reduce pain,2 lower blood pressure, and prevent cardiovascular diseases.5 To overcome these problems, finding new inhibitors from natural plants has been investigated. A few sEH inhibitors from natural products have been identified. The results showed that natural compounds found to inhibit sEH were diverse including biflavonoids,6 selaginellin,7 stilbenes,8 anthraquinone derivatives,9 carbazole-type alkaloids (isomahanine, bisisomahanine),10 alkylphloroglucinol derivatives, and triterpenoids.11 These results encourage us to continue our studies in discovery of sEH inhibitors from natural sources.

From our screening results, we found that the ethanol extract of Passiflora edulis Sims had appreciable inhibitory activity. Passiflora edulis Sims (Passifloraceae), a popular tropical fruit throughout the world, is usually used for juice production,12 and widely cultivated in South America, Africa, and Asia. In Vietnam, P. edulis is popularly cultivated in Tay Nguyen, Nghe An and Son La with areas of over ten thousand hectares. P. edulis was found to possess biological activities including anti-inflammatory,13 antihypertensive,14 anti-oxidant,15 anti-tumor,16 anti-anxiety,17 antifungal,18 and found to inhibit melanogenesis and promote collagen synthesis.19 Previous studies on chemical constituents of P. edulis showed the presence of triterpenoids,20 flavonoids,21 alkaloids,22 carotenoids,23 stilbenoids,1924 oil, and tocopherols.25 In spite of the number of studies that have been performed,192425 there has been no investigation of chemical constituents and sEH inhibitory activity of P. edulis seeds cultivated in Vietnam. Therefore, this paper described the isolation and structural elucidation of these compounds as well as the evaluation of their inhibitory activity on sEH.

After removing the oil (vegetable oil) from the seeds of P. edulis by n-hexane, the residue was extracted with ethanol to obtain ethanol extract. In the search for sEH inhibitors from natural sources, we found that the ethanol extract of the seeds of P. edulis inhibited 64.7% of sEH activity at a concentration of 37.5 µg/mL. This extract was then partitioned with dichloromethane (CH₂C_l2), ethyl acetate (EtOAc) fractions and aqueous residue. In the preliminary experiment, we tested the inhibitory activity of these fractions at 37.5, 75.0 and 150.0 µg/mL.

Based on the results in Table 1, CH₂C_l2- soluble fraction showed 91.8% inhibition at the concentration of 37.5 µg/mL, which was approximately 2.0-fold more potent than aqueous layer (48.7%). Interestingly, the EtOAc-soluble fraction exhibited very potently with > 100% sEH activity at the same concentration. Considering that EtOAc-soluble fraction showed the strongest action, our subsequent studies focused on the isolation of active components. This sub-fraction was subjected to column chromatography on a silica gel and C18-RP silica gel column to obtain five compounds (1 – 5) (Fig. 1).

Compound 1 was obtained as an ivory amorphous powder. The ¹H-NMR of 1 showed signals the presence of 1,3,5-trisubstituted benzene ring characterized with AB₂ system [δ_H 6.47 (2H, d, J = 2.0 Hz, H-2/H-6) and 6.18 (1H, t, J = 2.0 Hz, H-4)], and a 1,4-disubstituted benzene ring characterized with an A₂B₂ system [δ_H 7.36 (2H, d, J = 8.5 Hz, H-2′/H-6′) and 6.78 (2H, d, J = 8.5 Hz, H-3′/H-5′) together with trans-olefinic protons [δ_H 6.97 (1H, d, J = 16.0 Hz, H-8) and 6.81 (1H, d, J = 16.0 Hz, H-7)] (Fig. 1). The ¹³C-NMR spectrum of 1 exhibited 14 carbon signals including 12 aromatic carbons [δ_C 102.6 – 159.6], belonging to two benzene rings, and two olefinic carbons [δ_C 129.3 (C-7), and 128.7 (C-8)] (Fig. 1). The ESI-MS data of 1 indicated the pseudo molecular ion at m/z 229.09 for the [M+H]⁺, indicating a molecular weight of 228. Its molecular formula was determined to be C₁₄H₁₂O₃, according to ESI-MS, ¹H- and ¹³C-NMR spectroscopic data. Based on the above evidence and comparison with reported data,26 compound 1 was identified as trans-resveratrol. Compound 2, a derivative of 1, which the ¹H- and ¹³C-NMR spectra were similar to those of 1 except for the presence of 1,3,4-trisubstituted benzene ring characterized with ABX system [δ_H 7.07 (1H, d, J = 2.0 Hz, H-2′), 6.89 (1H, dd, J = 8.5, 2.0 Hz, H-6′), and 6.82 (1H, d, J = 8.5 Hz, H-5′)] in 2 (Fig. 1). The ESI-MS spectrum of compound 2 showed the pseudo molecular ion at m/z 245.08 [M+H]⁺, indicating the molecular formula C₁₄H₁₂O₄. Thus, compound 2 was identified as trans-piceatannol in comparison with literature data.27 These compounds (1 – 2) possess the basics of stilbene skeleton and are known to inhibit anti-oxidant, anti-inflammatory, anti-diabetes and anticancer.28

Compound 3 was obtained as yellow amorphous solid. The ¹H-NMR of 3 showed signals of 1,2,4-trisubstituted benzene rings characterized with two ABX systems [δ_H 7.63 (1H, d, J = 8.5 Hz, H-5), 7.54 (1H, d, J = 2.0 Hz, H-8), and 7.26 (1H, dd, J = 8.5, 2.0 Hz, H-6); δ_H 6.86 (1H, d, J = 8.5, Hz, H-5′), 6.73 (1H, d, J = 2.0 Hz, H-2′), and 6.72 (1H, dd, J = 8.5, 2.0 Hz, H-6′)], together with an olefinic proton at δ_H 6.71 (1H, s, H-2) (Fig. 1). The ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of 3 showed signals for six aromatic quaternary carbon [δ_C 169.8 (C-7), 168.4 (C-9), 149.3 (C-4′), 146.7 (C-3′), 125.5 (C-1′), 114.8 (C-10)] and six aromatic carbons [δ_C 126.8 (C-5), 126.3 (C-6′), 118.9 (C-2′), 116.7 (C-5′), 114.6 (C-6) and 99.3 (C-8)] (Fig. 1). Furthermore, a carbonyl carbon at δ_C 184.5 (C-4), together with two olefinic carbons [δ_C 147.7 (C-3), and 114.1 (C-2)] were also observed in the ¹³C NMR spectrum indicated that 3 was aurone skeleton (Fig. 1).29 The ESI-MS spectrum of compound 3 showed the pseudo molecular ion at m/z 271.06 [M+H]⁺, indicating the molecular formula C₁₅H₁₀O₅. Based on the above evidence and comparison with reported data,30 compound 3 was identified as sulfuretin, which was isolated from P. edulis for the first time. This compound possessed anti-inflammatory,31 anticancer,32 and neuroprotective activities.33

Compound 4 was isolated as yellow amorphous powder. The ¹H-NMR spectrum of 4 showed an aldehyde proton at δ_H 9.60 (1H, d, J = 8.0 Hz, H-9′), trans-olefinic protons at δ_H 6.70 (1H, dd, J = 15.5, 8.0 Hz, H-8′) and 7.61 (1H, d, J = 15.5 Hz, H-7′), which were assigned to a trans-cinnamaldehyde moiety. In addition, the ¹H-NMR of 4 showed two sets of aromatic protons signal [δ_H 6.97 (1H, s, H-2), 6.85 (1H, d, J = 8.0 Hz, H-5), 6.80 (1H, d, J = 8.0, H-6); δ_H 7.31 (1H, s, H-6′), 7.25 (1H, s, H-2′) arising from 1,3,4-trisubstituted and 1,3,4,5-tetrasubstituted benzene rings, respectively (Fig. 1). A stereochemistry of the dihydrobenzofuran ring [δ_H 5.63 (1H, d, J = 6.5 Hz, H-7), δ_H 3.59 (1H, q, J = 6.5 Hz, H-8) and δ_H 3.91-3.87 (2H, m, H-9)], and two singlets of methoxy groups [δ_H 3.92 (3H, s, 3′-OCH₃), and 3.84 (3H, s, 3-OCH₃)] were also observed in the ¹H-NMR spectrum. The large proton coupling constant between H-7 and H-8 (J_7,8 = 6.5 Hz) suggested the dihydrofuran ring has a trans-configuration. The ¹³C-NMR and DEPT spectra of 4 revealed the presence of twelve aromatic carbons, two olefinic carbons, an aldehyde carbon [δ_C 196.1 (C-9′)], a hydroxymethyl carbon [δ_C 64.5 (C-9)] and two methoxy carbons [δ_C 56.8 (3-OCH₃), and 56.4 (3′-OCH₃)] (Fig. 1). The HMBC correlations of proton H-2 at δ_H 6.97 (1H, s) and methoxy protons at δ_H 3.84 (3H, s) to carbon signal at δ_C 149.2 (C-3), as well as proton H-2′ at δ_H 7.25 (1H, s) and methoxy protons at δ_H 3.92 (3H, s) to carbon signal at δ_C 156.1 (C-3′), suggested that two methoxy groups were located at C-3 and C-3′ (Fig. 1). Further analysis of these signals by the COSY, HMQC and HMBC spectra led to the partial structures of 4. The ESI-MS data of 4 indicated the pseudo molecular ion at m/z 357.14 for the [M+H]⁺, indicating a molecular weight of 356. Its molecular formula was determined to be C₂₀H₂₀O₆, according to ESI-MS, ¹H- and ¹³C-NMR spectroscopic data. Therefore, compound 4 was identified as (+)-balanophonin,34 which was isolated from Passiflora genus for the first time. This compound was known to possess anti-oxidant,3536 anti-cholinesterase, 36 anti-inflammatory, anticancer, and anti-neurodegenerative activities.37

Compound 5 was isolated as brown amorphous powder. The ¹H-NMR spectrum of 5 showed the presence of two ABX system signals for the A ring [δ_H 6.68 (1H, d, J = 2.0 Hz, H-2′), 6.67 (1H, d, J = 8.5 Hz, H-5′), 6.50 (1H, dd, J = 8.5, 2.0 Hz, H-6′)] and the D ring [δ_H 7.15 (1H, d, J = 2.0 Hz, H-2), 7.09 (1H, dd, J = 8.5, 2.0 Hz, H-6), 6.96 (1H, d, J = 8.5 Hz, H-5)], two sets of AB₂ system signals for the B and C rings [6.48 (2H, overlap, H-12/H-12′), 6.19 (1H, t, J = 2.0 Hz, H-14′), 6.17 (1H, t, J = 2.0 Hz, H-10′), 6.12 (2H, d, J = 2.0 Hz, H-10/H-14)], two doublets for the trans-olefinic protons [δ_H 6.99 (1H, d, J = 16.0 Hz, H-7), 6.88 (1H, d, J = 16.0 Hz, H-8)], and two equivalent oxybenzyl methine protons [δ_H 4.75 (2H, d, J = 2.5 Hz, H-7′/H-8′)] (Fig. 1). The ¹³C-NMR and DEPT spectra of 5 exhibited 28 carbon signals including 24 aromatic carbons [δ_C 102.9 – 159.6], belonging to four benzene rings, and two olefinic carbons [δ_C 129.4 (C-7), and 128.4 (C-8)]. In addition, the carbon signals [δ_C 82.2 (C-8′) and 81.8 (C-7′)] indicate the presence of two equivalent oxybenzyl methine carbons. Analysis of these signals by the COSY, HMQC and HMBC spectra led to the partial structures of compound 5 (Fig. 1). The ESI-MS data of 5 indicated the pseudo molecular ion at m/z 487.13 for the [M+H]⁺, indicating a molecular weight of 486. Its molecular formula was determined to be C₂₈H₂₂O₈, according to ESI-MS, ¹H- and ¹³C-NMR spectroscopic data. The relative configuration between the C-7′ and the C-8′ positions was concluded to be cis from the coupling constant of the two oxybenzylmethine protons (J = 2.5 Hz). Based on the above evidence and comparison with reported data,38 compound 5 was identified as cassigarol E, which was isolated from Passiflora genus for the first time. Cassigarol E possessed anti-HIV-1,39 antidiabetic,40 and anticancer activities.41

The effect of isolated compounds (1 – 5) from P. edulis on sEH inhibitory activities was evaluated. The sEH inhibitory activities were determined using recombinant human sEH incubated with PHOME, an artificial substrate for fluorescence detection with AUDA (IC₅₀ value 4.4 nM), a sEH inhibitor as the positive control. The result showed that compound 4 had no inhibitory (N.I) effect on the activity of enzyme sEH at a concentration of 100 µM, while compounds 1 and 2 at a concentration of 100 µM exhibited the highest sEH inhibitory activity (>100%) (Table 1). Sulfuretin (3), and cassigarol E (5) also showed a strong inhibitory effect on sEH with their inhibition values in the range of 74.6 and 77.7%, respectively (Table 1). Compounds 1 – 3, and 5 showed inhibitory rates over 50% and were further evaluated at concentrations ranging from 6.2 to 100 µM, to elucidate the IC₅₀ values. These inhibitors (1 – 3, and 5) showed dose-dependent inhibition, with IC₅₀ values of 14.2 ± 0.6, 3.4 ± 4.8, 15.8 ± 1.0, and 14.4 ± 0.8 µM, respectively (Table 2).

Previously, natural plant components with stilbene skeletons from other plants have also shown inhibitory effects on sEH activity. Rhapontigenin, isorhapontin and astringin from Rheum undulatum (Polygonaceae),8 and 2-isopropyl-5-[(E)-2- phenylvinyl]benzene-1,3-diol have displayed potent sEH inhibitory activity.42 Several stilbene from Polygonum multiflorum (Polygonaceae), such as (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside, (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-xyloside, and (E)-2,3,5,4′-tetrahydroxystilbene-2-O-β-D-(6′′-O-acetyl)-glucoside completely inhibited sEH in a dose-dependent manner, with low IC₅₀ values.43 As in this study, some isolated stilbenes from P. edulis can manifest sEH inhibitory activity. Such compounds have been purified from natural medicinal plants for many years all over the world. In addition to other medicinal properties, such as anti-inflammatory, anticancer, and antioxidant activities, the sEH inhibitory activity is worthy of notice, because low-molecular-weight materials can easily reach the site of action following oral administration since they cross the blood-brain barrier.44