Occurrence of urea-based soluble epoxide hydrolase inhibitors from the plants in the order Brassicales

RESEARCH ARTICLE

Occurrence of urea-based soluble epoxide

hydrolase inhibitors from the plants in the

order Brassicales

Seiya Kitamura, Christophe Morisseau, Todd R. Harris, Bora Inceoglu, Bruce

D. Hammock*

Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center, University of

California Davis, Davis, California, United States of America

a 1111111111

* bdhammock@ucdavis.edu

Abstract

Recently, dibenzylurea-based potent soluble epoxide hydrolase (sEH) inhibitors were identi-

fied in Pentadiplandra brazzeana, a plant in the order Brassicales. In an effort to generalize

the concept, we hypothesized that plants that produce benzyl glucosinolates and corre-

sponding isothiocyanates also produce these dibenzylurea derivatives. Our overall aim here

was to examine the occurrence of urea derivatives in Brassicales, hoping to find biologically

active urea derivatives from plants. First, plants in the order Brassicales were analyzed for

the presence of 1, 3-dibenzylurea (compound 1), showing that three additional plants in the

order Brassicales produce the urea derivatives. Based on the hypothesis, three dibenzy-

lurea derivatives with sEH inhibitory activity were isolated from maca (Lepidium meyenii)

roots. Topical application of one of the identified compounds (compound 3, human sEH IC₅₀

= 222 nM) effectively reduced pain in rat inflammatory pain model, and this compound was

bioavailable after oral administration in mice. The biosynthetic pathway of these urea deriva-

tives was investigated using papaya (Carica papaya) seed as a model system. Finally, a

small collection of plants from the Brassicales order was grown, collected, extracted and

screened for sEH inhibitory activity. Results show that several plants of the Brassicales

order could be potential sources of urea-based sEH inhibitors.

OPEN ACCESS

Citation: Kitamura S, Morisseau C, Harris TR,

Inceoglu B, Hammock BD (2017) Occurrence of

urea-based soluble epoxide hydrolase inhibitors

from the plants in the order Brassicales. PLoS ONE

12(5): e0176571. https://doi.org/10.1371/journal.

pone.0176571

Editor: Luca Vanella, Universita degli Studi di

Catania, ITALY

Received: October 24, 2016

Accepted: April 12, 2017

Published: May 4, 2017

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files.

Introduction

Soluble epoxide hydrolase (sEH, EC 3.3.2.10) is the major enzyme responsible for the hydroly-

sis of epoxy fatty acids to their corresponding diols in humans and other mammals [1]. These

epoxy fatty acids are pleiotropic endogenous mediators with key functions in inflammation,

pain and blood pressure regulation [1–3]. Increasing the levels of endogenous epoxy fatty

acids by inhibiting sEH has been shown to block and resolve inflammation, reduce pain and

lower blood pressure in numerous in vivo animal models [2,4–9]. The 1, 3-disubstituted urea

moiety is known as a pharmacophore of many potent sEH inhibitors, in which the urea mim-

ics both the epoxide substrate and the transition state of epoxide hydrolysis, leading to

Funding: This work was partially funded by

National Institute of Environmental Health Sciences

(NIEHS, http://www.niehs.nih.gov/) grant

R01ES002710, and NIEHS Superfund Research

Program grant P42 ES04699. The content is solely

the responsibility of the authors and does not

necessarily represent the official views of the NIH.

SK was financially supported by Japan Student

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Plant sEH inhibitors

Services Organization (http://www.jasso.go.jp/).

The funders had no role in study design, data

collection and analysis, decision to publish, or

preparation of the manuscript.

competitive inhibition of sEH [10–13]. Several groups are working to move sEH inhibitors to

the clinic for treating human and equine disorders [14]. So far these compounds appear to

have a large therapeutic index and thus provide an excellent margin of safety [1]. However,

this traditional process of drug development takes many years, and none of sEH inhibitors are

in clinical use yet. Alternatively, sEH inhibitors derived from natural products, especially edi-

ble vegetables, could provide a shorter path to treating patients and companion animals, offer-

ing inexpensive therapeutics to patients that will not require the same regulatory barriers as

pharmaceuticals [15,16]. In addition, study of these natural products will explain the modes of

action of some natural remedies.

Competing interests: The authors do not have a

direct financial interest or a patent directly related

to material and data described in this article.

Nevertheless, authors BI and BDH are cofounders

of Eicosis LLC, which is developing a chemical sEH

inhibitor for pharmacological used. Authors BI, CM

and BDH are coinventors on patents related to sEH

inhibitors filed and owned by the University of

California. BDH has more than 100 patents and

pending patent applications on various aspects of

research. An extensive list is publicly available at

the USPTO website. No patent has been filed

related to the materials described in this

Tsopmo et al. reported 1, 3-dibenzylurea derivatives in the root of a plant in Cameroon,

Pentadiplandra brazzeana, and we recently reported these urea derivatives as potent sEH

inhibitors [17,18]. A few others also reported benzylurea derivatives in plants [19–21]. Inter-

estingly, all of these plants are members of the order Brassicales. These plants were also

reported to produce benzyl glucosinolates or benzyl isothiocyanates (chemical structures

shown in Fig 1) [22–25]. Glucosinolates and isothiocyanates are well-recognized secondary

metabolites of plants in the order Brassicales, while urea derivatives in nature have not been

studied extensively. We hypothesized that some of the plants that produce benzyl glucosino-

lates and benzyl isothiocyanates also produce these dibenzylurea derivatives.

manuscript, nor on any closely related research.

This does not alter the authors’ adherence to all the

PLOS ONE policies on sharing data and materials.

Our overall aim was to examine the occurrence of urea derivatives in Brassicales, hoping to

find biologically active urea-based sEH inhibitors from plants. To accomplish this, we looked

for 1, 3-dibenzylurea (compound 1) in plants in the order Brassicales that have been reported

to produce benzyl glucosinolate or benzyl isothiocyanate. Next, meta methoxy substituted ben-

zylurea derivatives, which were predicted based on the hypothesis, were isolated from maca

(Lepidium meyenii) root and assayed for sEH inhibitory potency in vitro. The most potent

compound (compound 3) showed in vivo analgesic effects in a rat inflammatory pain model,

and was bioavailable after oral administration. Possible biosynthetic pathways of compound 1

were studied using papaya seed as a model system. Finally, a small collection of plants from the

Brassicales order was grown, collected, extracted and screened for sEH inhibitory activity and

for the occurrence of urea derivatives.

Materials and methods

General experimental procedures

All reagents and solvents were purchased from commercial suppliers and were used without

further purification. All reactions were performed in an inert atmosphere of dry nitrogen or

argon. UV absorption spectra were measured on a Varian Cary 100 Bio UV-Visible Spectro-

photometer. Melting points were determined using an OptiMelt melting point apparatus.

NMR spectra were collected using a Varian 400 or 600 MHz, or Bruker Avance III 800 MHz

spectrometer with chemical shifts reported relative to residual deuterated solvent peaks or a

tetramethylsilane internal standard. Accurate masses were measured using a LTQ orbitrap

hybrid mass spectrometer or Micromass LCT ESI-TOF-MS. FT-IR spectra were recorded on a

Thermo Scientific NICOLET IR100 FT-IR spectrometer. The purity of all synthetic com-

pounds were found to be > 95% based on NMR analysis. The purity of the compounds that

were tested in the assay were further determined by reverse phase HPLC-DAD and found to

be > 95% at 254 nm absorption (LC method detailed in S3 Table).

Plant samples

The plant species were authenticated by a botanist Dr. Ellen Dean at UC Davis Center for

Plant Diversity, where a voucher specimen of papaya (Carica papaya) fruit (DAV211613),

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Plant sEH inhibitors

Fig 1. Chemical structures of 1, 3-dibenzylurea, benzyl glucosinolate and isothiocyante.

https://doi.org/10.1371/journal.pone.0176571.g001

papaya seed (DAV211614), garden cress (Lepidium sativum, DAV211612, DAV211953), and

water cress (Nasturtium officinale, DAV216312) were deposited. Fresh papaya fruit imported

from Mexico was purchased at La Superior Supermercados (Woodland, CA) between Nov.

2014 and Apr. 2015. Fresh water cress produced in California was purchased at Nugget Market

(Davis, CA) between Feb. 2016 and Aug. 2016. Garden cress and water cress seeds were pur-

chased from Botanical Interests Inc. and germinated following the manufacturer’s protocol.

The whole sprouts were harvested 2 weeks after seeding between Dec. 2014 and Aug. 2016.

Maca dried root powder imported from Peru was purchased from Herbs America (100% Raw

Maca Powder, product name ‘Maca Magic™’). The organism of origin was specified by DNA

sequencing, and is included in the Supporting information (S1 Text). The powder specimen

was deposited at the Center for Plant Diversity at UC Davis (DAV211952).

Extraction for LC-MS/MS analysis

Fresh papaya seeds (2 g) or fresh garden cress sprouts (300 mg) were ground and incubated

overnight at RT, then extracted with dichloromethane (DCM)-methanol (MeOH) (1:1) by

sonication (2 x 3 mL) at RT (5 min per extraction). The dried root powder of maca (100 mg)

was extracted with the same method. The extracts were concentrated in vacuo, reconstituted in

MeOH and filtered, then diluted to the appropriate concentration for the LC-MS/MS analysis.

Standard addition and recovery experiments were performed as described previously [17].

LC-MS/MS analysis

Compounds were analyzed using a Waters Quattro Premier triple quadrupole tandem mass

spectrometer (Micromass, Manchester, UK) interfaced to an electrospray ionization (ESI)

source. The ESI was performed following HPLC in the positive mode at 2.51 kV capillary volt-

age. The source and the desolvation temperatures were set at 120 and 300˚C, respectively.

Cone gas (N₂) and desolvation gas (N₂) were maintained at flow rates of 10 and 700 L/h,

respectively. Optimized conditions for mass spectrometry are shown in S1 Table. Dwell time

was set to 0.1 s. A regression curve for each compound was obtained from at least six different

concentrations of standard stock solutions (R²> 0.99). 1, 3-Diphenylurea was used as an

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internal standard and was added just before the analysis. The final concentration of 1, 3-diphe-

nylurea was adjusted to 100 nM.

The MS was coupled with a Waters Acquity UPLC (Waters, Milford, MA, USA). A Varian

Pursuit5 C18 RP HPLC column (150 mm × 2.1 mm, particle size 5 μm) was used to separate

the analytes. The HPLC solvent gradient is shown in S3 Table.

Isolation and purification of urea derivatives

The dried root powder of maca (20 kg) was extracted with DCM-MeOH (1:1) by shaking (3 x

40 L) at room temperature (at least 120 min per shaking). The combined extracts were concen-

trated in vacuo, and the residue was partitioned between DCM and brine. Concentrating the

DCM layer in vacuo yielded the crude extract (612 g) as a dark brown oil. Flash column chro-

matography on a Si gel column eluting with hexane: ethyl acetate (1:1) or DCM: MeOH (30:1

or 50:1) was repeated, followed by repetitive preparative scale normal phase HPLC (Phenom-

enex Luna Silica (2) column, 250 × 21.2 mm, 5 μm, Waters ELSD 2424 evaporative light scat-

tering detector and 1525 Binary HPLC Pump) eluting with hexane: isopropanol (9:1) at a flow

rate of 20 mL/min. Recrystallization from DCM/hexane afforded compound 1 (31 mg) and

compound 2 (36 mg). Further purification by reverse phase HPLC (Phenomenex Luna C18

(2) column, 250 × 21.2 mm, 5 μm) eluting with water: MeOH (50–80% gradient in 20 min, 12

mL/min) followed by a short flash column chromatography on a Si gel eluting with DCM:

MeOH (30:1) afforded compound 3 (1.5 mg). It should be noted that dibenzyl thioureas were

not observed in dried maca root powder. Therefore, it is unlikely that urea derivatives in maca

root were produced during the extraction and purification.

1, 3-Dibenzylurea (compound 1): off-white powder (DCM); mp 166–170˚C (lit.[18] 168–

170˚C); UV (acetonitrile) λ_max(log ε): 258 (2.26) nm; IR (neat) ν_max3321, 1626, 1572, 1493,

1453, 1254, 752 cm^-1; ¹H NMR (800 MHz, DMSO-d₆) δ 7.31 (t, J = 7.6 Hz, 4H, H-5, H-7), 7.25

(d, J = 6.7 Hz, 4H, H-4, H-8), 7.22 (t, J = 7.2 Hz, 2H, H-6), 6.44 (s, 2H, NH), 4.23 (d, J = 6.0 Hz,

4H, H-2). ¹³C: NMR (201 MHz, DMSO-d₆) δ 158.1 (C, C-1), 140.9 (C, C-3), 128.2 (CH, C-5,

C-7), 127.0 (CH, C-4, C-8), 126.6 (CH, C-6), 43.0 (CH₂, C-2). HRESIMS m/z 241.1336 (S4 Fig

Calculated for [C₁₅H₁₇N₂O]⁺, 241.1335).

1-Benzyl-3-(3-methoxybenzyl) urea (compound 2): off-white powder (DCM); mp 101–

107˚C (synthetic standard (acetone) 108.3–109.1 (108.6˚C); UV (acetonitrile) λ_max(log ε): 272

(3.25) nm; IR (neat) ν_max3349, 3317, 3032, 2923, 1625, 1577, 1511, 1242, 1031 cm^-1; ¹H and ¹³

NMR see Fig 2. HRESIMS m/z 271.1441 (S5 Fig Calculated for [C₁₆H₁₉N₂O₂]⁺, 271.1441).

1, 3-Bis (3-methoxybenzyl) urea (compound 3): off-white solid; UV (acetonitrile) λ_max

(log ε): 273 (3.31) nm; IR (neat) ν_max3321, 1621, 1589, 1574, 1488, 1472, 1435, 1296, 1263,

1155, 1049, 785 cm^-1; ¹H and ¹³C NMR see Fig 2. HRESIMS m/z 301.1540 (S6 Fig Calculated

for [C₁₇H₂₁N₂O₃]⁺, 301.1546).

C

Synthesis of ureas and thioureas

Compound 1, 1-(adamantan-1-yl)-3-(5-(2-(2-ethoxyethoxy) ethoxy) pentyl) urea (AEPU),

and 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) were previously

synthesized [12,26,27].

General procedure of urea and thiourea synthesis. An amine (1 equiv) was added to a

solution of benzyl isocyanate or benzyl isothiocyanate in THF. After stirring for 10 min at

room temperature, hexane was added and the resulting white crystals were collected. Recrys-

tallization from acetone was repeated until the target compound was > 95% pure as judged by

NMR analysis.

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Fig 2. NMR spectroscopic data (¹H 800 MHz, ¹³C 201 MHz) for compound 2 (DMSO-d₆) and compound 3

(CDCl₃).

https://doi.org/10.1371/journal.pone.0176571.g002

1-Benzyl-3-(3-methoxybenzyl) urea (compound 2): off-white powder (260 mg, 0.963

mmol, 75%); mp 108.3–109.1 (108.6˚C; ¹H and ¹³C NMR: identical to compound 2 isolated

from maca (Fig 2); ESI-MS [M+Na]⁺m/z 293.11 (calculated for C₁₆H₁₈N₂NaO₂293.13),

Purity > 99% (HPLC-UV (254 nm), ^tR = 9.18 min).

1, 3-Bis (3-methoxybenzyl) urea (compound 3): white powder (45 mg, 0.15 mmol, 15%);

mp 112.6–113.8 (113.0˚C; ¹H and ¹³C NMR: identical to compound 3 isolated from maca (Fig

2); ESI-MS [M+Na]⁺m/z 323.11 (calculated for C₁₇H₂₀N₂NaO₃323.14), Purity > 99%

(HPLC-UV (254 nm), ^tR = 9.01 min).

1-Benzyl-3-(4-isopropylphenyl) urea (compound 4): white powder (20 mg, 75 μmol, 4%);

mp 111.8–112.2 (112.0˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 8.43 (s, 1H), 7.34–7.29 (m, 6H),

7.25–7.22 (m, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.54 (t, J = 6.0 Hz, 1H), 4.28 (d, J = 5.9 Hz, 2H),

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2.79 (hept, J = 7.0 Hz, 1H), 1.16 (d, J = 6.9 Hz, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 155.3,

141.1, 140.4, 138.2, 128.3, 127.1, 126.7, 126.3, 117.9, 42.7, 32.7, 24.1. ESI-MS [M+Na]⁺m/z

291.15 (calculated for C₁₇H₂₀N₂NaO 291.15).

1-Benzyl-3-(4-isopropylphenyl) thiourea (compound 5): white powder (109 mg,

384 μmol, 38%) mp 110.0–110.3 (110.1˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 9.53 (s, 1H), 8.08

(s, 1H), 7.34–7.19 (m, 9H), 4.73 (d, J = 5.8 Hz, 2H), 2.86 (hept, J = 6.9 Hz, 1H), 1.19 (d, J = 6.9

Hz, 6H).¹³C NMR (151 MHz, DMSO-d₆) δ 180.7, 144.6, 139.2, 136.7, 128.2, 127.4, 126.8, 126.5,

123.7, 47.2, 33.0, 23.9. ESI-MS [M+Na]⁺m/z 307.10 (calculated for C₁₇H₂₀N₂NaS 307.12).

1, 3-Diphenethylurea (compound 8): white powder (15 mg, 56 μmol, 3%) mp 138.5–138.9

(138.8˚C;¹H NMR (600 MHz, DMSO-d₆) δ 7.29 (t, J = 7.5 Hz, 4H), 7.19 (d, J = 8.2 Hz, 4H),

5.87 (t, J = 5.8 Hz, 2H), 3.21 (m, 4H), 2.66 (t, J = 7.2 Hz, 4H). ¹³C NMR (151 MHz, DMSO-d₆)

δ 157.8, 139.7, 128.6, 128.2, 125.9, 40.8, 36.14. HRESI-MS [M+H]⁺m/z 269.1649 (calculated

for C₁₇H₂₁N₂O 269.1648). Purity 98% (HPLC-UV (254 nm), ^tR = 10.12 min).

1, 3-Diphenethylthiourea (compound 9): off-white crystals (219 mg, 771 μmol, 39%) mp 93–

96 (94˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 7.45 (s, 2H), 7.30 (t, J = 7.6 Hz, 4H), 7.25–7.16 (m,

6H), 3.59 (s, 4H), 2.79 (t, J = 7.6 Hz, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 182.1, 139.4, 128.6,

128.3, 126.1, 44.9, 40.1, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 34.8. HRESI-MS [M+H]⁺m/z 285.1422

(calculated for C₁₇H₂₁N₂S 285.1424). Purity 98% (HPLC-UV (254 nm), ^tR = 11.45 min).

1-(4-Hydroxyphenethyl)-3-phenethylurea (compound 10): white powder (135 mg,

475 μmol, 48%) mp 157–159 (158˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 9.14 (s, 1H), 7.29 (t,

J = 7.5 Hz, 2H), 7.22–7.17 (m, 3H), 6.97 (dd, J = 8.7, 2.7 Hz, 2H), 6.70–6.64 (m, 2H), 5.83 (dt,

J = 27.7, 5.8 Hz, 2H), 3.24–3.18 (m, 2H), 3.14 (q, J = 6.5 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.54

(t, J = 7.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 157.8, 155.5, 139.7, 129.7, 129.4, 128.6,

128.2, 125.9, 115.0, 41.2, 40.8, 36.2, 35.3. HRESI-MS [M+H]⁺m/z 285.1596 (calculated for

C₁₇H₂₁N₂O₂285.1598). Purity >99% (HPLC-UV (254 nm), ^tR = 8.72 min).

1-(4-Methoxybenzyl)-3-phenethylurea (compound 11): white powder (303 mg, 475 μmol,

53%) mp 122.8–123.4 (123.1˚C; ¹H NMR (400 MHz, DMSO-d₆) δ 7.33–7.24 (m, 2H), 7.24–

7.11 (m, 5H), 6.91–6.83 (m, 2H), 6.26 (t, J = 5.9 Hz, 1H), 5.88 (t, J = 5.8 Hz, 1H), 4.12 (d, J = 5.9

Hz, 2H), 3.72 (s, 3H), 3.30–3.19 (m, 2H), 2.68 (t, J = 7.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 158.0, 157.9, 139.7, 132.8, 128.7, 128.3, 128.3, 125.9, 113.6, 55.0, 42.3, 41.0, 36.2. HRE-

SI-MS [M+H]⁺m/z 285.1597 (calculated for C₁₇H₂₁N₂O₂285.1598). Purity 99% (HPLC-UV

(254 nm), ^tR = 9.69 min).

General procedure of chemical demethylation

To a solution of compound 3 in dichloromethane, 1 M tribromoborane (1.2 equiv or 3.0

equiv) solution in DCM was added dropwise at -78˚C. The reaction was slowly warmed up to

RT, and stirred for 24 h. To this solution was added dropwise water at 0˚C, then warmed to

RT. To this solution was added ethyl acetate, extracted, and washed with brine twice. The

organic layer was dried over MgSO₄, filtered and concentrated in vacuo. The target compound

was purified by column chromatography followed by recrystallization from DCM.

1-(3-Hydroxybenzyl)-3-(3-methoxybenzyl) urea (compound 6): white powder (5 mg,

17 μmol, 5%) mp 124.8–125.6 (125.3˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 9.30 (s, 1H), 7.22

(t, J = 7.8 Hz, 1H), 7.08 (t, J = 7.7 Hz, 1H), 6.83–6.81 (m, 2H), 6.78 (dd, J = 8.5, 2.6 Hz, 1H),

6.66 (d, J = 8.3 Hz, 2H), 6.61–6.59 (m, 1H), 6.38 (dt, J = 11.7, 6.1 Hz, 2H), 4.20 (d, J = 6.0 Hz,

2H), 4.14 (d, J = 6.0 Hz, 2H), 3.72 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆) δ 159.7, 158.4,

157.8, 143.0, 142.8, 129.7, 129.6, 119.6, 118.0, 114.3, 113.9, 112.9, 112.4, 55.4, 43.3 (x2).

ESI-MS [M+H]⁺m/z 287.12 (calculated for C₁₆H₁₉N₂O₃287.14), Purity >99% (HPLC-UV

(254 nm), ^tR = 7.87 min).

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1, 3-Bis(3-hydroxybenzyl)urea (compound 7): white powder (58 mg, 213 μmol, 64%) mp

155.3–157.0 (156.1˚C; ¹H NMR (600 MHz, DMSO-d₆) δ 9.30 (s, 2H), 7.08 (t, J = 8.0 Hz, 2H),

6.67–6.65 (m, 4H), 6.60 (ddd, J = 8.0, 2.3, 1.2 Hz, 2H), 6.33 (t, J = 6.0 Hz, 2H), 4.14 (d, J = 6.0

Hz, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 158.0, 157.3, 142.3, 129.2, 117.6, 113.9, 113.5, 42.9.

ESI-MS [M+H]⁺m/z 273.13 (calculated for C₁₅H₁₇N₂O₃273.12), Purity 95% (HPLC-UV (254

nm), ^tR = 5.81 min).

Variable incubation conditions and trapping experiments

For each of the conditions, extraction and incubation was performed as described above. For

the conditions without overnight incubation, DCM/MeOH (1:1) was added immediately after

grinding the plant material and then extracted. For methanol treatment, MeOH (2 mL) was

added and then ground and incubated at RT overnight. For microwave heating, 2 g of papaya

seeds were put in a sealed tube and heated to 100˚C for 20 min using a high-pressure micro-

wave digestion unit (Ethos SEL. Milestone, Italy). After cooling to RT, seeds were ground and

incubated at RT overnight. For liquid nitrogen treatment, before grinding, fresh papaya seeds

were frozen by the addition of liquid nitrogen (approximately 5 mL), then ground and

extracted with DCM/MeOH (1:1) without incubation. For the trapping experiments, 2 mL of

an aqueous solution (containing 1% v/v DMSO) of 4-isopropylaniline (10 μM) or compound 5

(10 μM) was added to fresh papaya seeds (2 g). This was ground thoroughly and incubated

overnight at RT.

Enzyme purification

Recombinant sEH was produced in insect High Five cells using recombinant baculovirus

expression vectors, and purified by affinity chromatography as reported previously [28,29].

The enzyme appeared as a single band (0.3 μg loading) with an estimated purity of more than

95% by Coomassie Brilliant Blue staining following SDS-PAGE separation. The final recombi-

nant sEH preparations had no esterase or glutathione S-transferase activities, which interfere

with the CMNPC assay as described below.

Measurement of sEH inhibition by fluorescent assay (CMNPC assay)

IC₅₀values were determined as described previously [30] using cyano (2-methoxynaphthalen-

6-yl) methyl trans-(3-phenyl-oxyran-2-yl) methyl carbonate (CMNPC) as a fluorescent sub-

strate. Recombinant sEH (0.96 nM) was incubated with inhibitors for 5 min in 25 mM bis-

Tris/HCl buffer (pH 7.0) containing 0.1 mg/mL of BSA at 30˚C prior to substrate introduction

([S] = 5 μM). Activity was measured by determining the appearance of the 6-methoxy-

2-naphthaldehyde with an excitation wavelength of 330 nm and an emission wavelength of

465 nm for 10 min.

Ethics statement

All of the animal experiments were conducted in line with federal regulations and were per-

formed according to protocols approved by the Animal Use and Care Committee of the Uni-

versity of California, Davis.

Nociceptive assay using the rat inflammatory pain model

Male Sprague-Dawley rats weighing 225–250 g were obtained from Charles River Laboratories

and maintained in the UC Davis animal housing facility with ad libitum water and food on a

12 h/12 h light-dark cycle. Behavioral nociceptive testing was conducted by assessing

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Plant sEH inhibitors

mechanical withdrawal threshold using an electronic von Frey anesthesiometer apparatus

(IITC, Woodland Hills, CA) as described previously [31]. The analgesic effect of compound 3

was tested using the intraplantar carrageenan-induced local inflammatory pain model [12,31].

Following baseline measurements, carrageenan (50 μL, 1% solution of carrageenan in ddH₂O)

was administered into the plantar area of one hind paw. At 4 h post administration of carra-

geenan, responses were measured, and immediately after these measurement, 3 (3% in

PEG400/Vanicream™ (Pharmaceutical Specialties, Inc. Rochester, MN) = 3:7, 6 mg of com-

pound/paw), the synthetic sEH inhibitor triclocarban (TCC, 3%), or the vehicle (200 μL of

PEG400/Vanicream™) was administered onto the inflamed paw by topical application [4]. The

cream was thoroughly massaged across the entire hind paw surface over a 1–2 min period.

Subsequently, the ability of these treatments to reduce the carrageenan-induced inflammatory

pain was monitored over the course of 4 h.

Statistical analysis

Data were analyzed using SigmaPlot 11.0 for Windows (Systat Software Inc., San Jose, CA).

Kruskal-Wallis One Way ANOVA on Ranks followed by Tukey Test was performed with p

values < 0.05 considered significant.

Pharmacokinetics

Pharmacokinetic profiles of the inhibitors were determined by following the procedure

described previously in literature [32,33] with some modifications. Male Swiss Webster mice

(7 weeks old, 24–30 g) were purchased from Charles River Laboratories. Inhibitors were dis-

solved in polyethylene glycol (average molecular weight: 300) to give a clear solution for oral

administration. Compounds were orally administered by gavage to mice at a dose of 10 mg/kg

(compound 3 & AEPU) and 0.3 mg/kg (TPPU) in 100–140 μL of vehicle depending on animal

weight. Blood (10 μL) was collected from the tail vein using a pipet tip rinsed with EDTA. The

blood samples were prepared according to the methods detailed previously [32], and blood

samples were analyzed by LC-MS/MS.

Brassicales library construction

Plant samples and their sources are listed in supporting information (S4 Table). The whole

sprouts were harvested 1–2 weeks after seeding depending on the growth of the sprouts.

Approximately 1 g of whole sprouts were ground and incubated at RT for 2 days, and extracted

as described above. Extract was reconstituted in DMSO (10 mg/mL), and IC₅₀was measured

using the fluorescent-based assay using CMNPC as a substrate. For MS screening, extracts

were diluted 100-fold in methanol containing 100 nM AUDA (internal standard, 12-(3-ada-

mantan-1-yl-ureido) dodecanoic acid), filtered, and analyzed for urea derivatives listed in the

supporting information (S2 Table).

Results and discussion

Occurrence and quantification of 1, 3-dibenzylureas in Brassicales

Plants in the order Brassicales, including maca (Lepidium meyenii) root, papaya (Carica

papaya) seed, and garden cress (Lepidium sativum) sprout, were tested for the presence of

compound 1 (1, 3-dibenzylurea) using LC-MS/MS. These plant species and parts were selected

based on previous reports of the presence of benzyl glucosinolate (glucotropaeolin) or benzyl

isothiocyanate [23,34,35]. Compound 1 was detected in all three plants, suggesting that the

occurrence of urea derivatives is conserved among these plant species (Table 1). To test the

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Plant sEH inhibitors

Table 1. Concentration of 1, 3-dibenzylurea (compound 1) in plant samples.

Concentration of compound 1

ng/g fresh weight ng/g dry weight

Papaya seed

Garden cress sprout

56 ± 11

370 ± 73

11,800 ± 1,800

6,900 ± 125

1,900 ± 200

250 ± 38

Maca dried root powder

Pentadiplandra brazzeana dried root powder^a

-

Mean ± SD values are shown.

^aData from Kitamura et al., (2015).

https://doi.org/10.1371/journal.pone.0176571.t001

efficiency of our extraction method, synthetic standard of compound 1 was spiked into the

plant samples. The recovery percentages were above 88%, suggesting that our extraction

method yields sufficient compound 1 from the plant samples.

Maca root has been reported to contain meta methoxy substituted benzyl isothiocyanate in

addition to non-substituted benzyl isothiocyanate [36,37], so we expected to find meta meth-

oxy substituted benzylureas. To our knowledge, there are no reports of dibenzylurea deriva-

tives from maca. Compound 1, unsymmetric 1-benzyl-3-(3-methoxybenzyl) urea (compound

2), and 1, 3-bis (3-methoxybenzyl) urea (compound 3) were isolated from maca root, and the

NMR data are shown in Figs 2 & 3 and S1–S3 Figs. All of the spectroscopic data are identical

to the synthetic standards, supporting the structure of these urea compounds (S1–S3 Figs).

Compound 2 has not been reported previously, while there is one report of compound 3

(called salvadourea) found in the plant Salvadora persica [19], a Brassicales species.

Soluble epoxide hydrolase inhibitory potency and physical properties of

dibenzylurea derivatives found in Brassicales

The sEH inhibitory potency of benzylurea derivatives found in Brassicales was measured in

order to understand their possible biological implications. As shown in Fig 4, compound 3 dis-

played inhibitory potency on human sEH with an IC₅₀value in the range of 200 nM, followed

by compound 2 with approximately 2-fold lower potency. Compound 3 is approximately

2-fold less potent than the para methoxy substituted benzylurea derivatives (MMU) found in

P. brazzeana [17,18], while the solubility of compound 3 is approximately 10-fold higher than

MMU. These are among the most potent sEH inhibitors derived from natural sources,

although they are still at least 100-fold less potent than highly potent synthetic inhibitors such

as trans-4-(4-(3-(4-trifluoromethoxy-phenyl)-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB)

which has an IC₅₀below 1 nM on the human sEH [38].

Brassicales-derived sEH inhibitor compound 3 effectively blocks

inflammatory pain

Several studies have shown that sEH inhibitors effectively reduce inflammatory and neuro-

pathic pain [4,5,7,12]. In order to evaluate the possible in vivo efficacy of plant urea derivatives,

a rat inflammatory pain model was used and the analgesic effects of compound 3 were deter-

mined. Compound 3 was selected based on its inhibitory potency on sEH and its solubility. As

shown in Fig 5, formulation in Vanicream and topical administration of either compound 3 (6

mg/paw) or the synthetic sEH inhibitor triclocarban (TCC, 6 mg/paw, positive control) signifi-

cantly reduced carrageenan induced inflammatory pain (Kruskal-Wallis One Way ANOVA

on Ranks, p ꢀ 0.001). TCC is an antimicrobial agent widely used in personal care products,

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Plant sEH inhibitors

Fig 3. Key HMBC and ¹H-¹H COSY correlations of compound 2 and 3.

https://doi.org/10.1371/journal.pone.0176571.g003

and is a potent synthetic sEH inhibitor with anti-inflammatory effects [39]. TCC possesses an

IC₅₀of 27 nM on rat sEH [40], which is approximately 6-fold more potent than compound 3.

As expected, TCC showed slightly higher efficacy than compound 3 (Tukey’s post hoc test,

compound 3 vs TCC, p < 0.05). The blood concentration of compound 3 was monitored dur-

ing the assay (Fig 5B). At 30 min the concentration peaked, reaching a level higher than

40 μM, suggesting that compound 3 had high permeability through skin. Although the con-

centration rapidly decreased, even after 4 hours, compound 3 was detected at concentrations

higher than 3 μM. It should be noted that local concentration of the compound in the inflamed

paw could be higher than systemic concentration. A brief time lag between the compound

concentration peak and the analgesic response was observed. This is consistent with earlier

observations when an sEH inhibitors were administered topically [4].

Brassicales-derived sEH inhibitor compound 3 is orally available

In order to determine pharmacokinetic properties, compound 3 was orally administered into

mice. As shown in Fig 6, compound 3 was rapidly absorbed following oral administration.

This result suggests that compound 3 could be absorbed when maca root powder is orally

taken. It should be noted that maca contains various other ingredients which may interfere

with absorption/distribution/metabolism, thus the pharmacokinetic profile of maca powder

could be significantly different from our results. Two synthetic sEH inhibitors, 1-(adamantan-

1-yl)-3-(5-(2-(2-ethoxyethoxy) ethoxy) pentyl) urea (AEPU) [27,41] and 1-trifluoromethoxy-

phenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) [12] were used as reference compounds.

AEPU is an early-generation synthetic sEH inhibitor which showed efficacy on the

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Plant sEH inhibitors

Fig 4. sEH inhibition potency of ureas found in Brassicales and related compounds.

https://doi.org/10.1371/journal.pone.0176571.g004

development of atherosclerosis in a mouse model when delivered in drinking water [42]. As

shown in Fig 6, the oral availability of compound 3 is higher than that of AEPU, while the

more recently developed synthetic sEH inhibitor, TPPU is more stable and has much higher

bioavailability than compound 3. Compound 3 has relatively high solubility (Fig 4), and thus

this compound may have better absorption compared to other urea derivatives found in Bras-

sicales. The mono-demethoxy metabolite of compound 3 (compound 6) was detected at simi-

lar concentrations to compound 3. Based on its concentration and potency (human sEH IC₅₀

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Plant sEH inhibitors

Fig 5. Topical treatment of compound 3 effectively reduces carrageenan-induced inflammatory pain

in rat. (A) Carrageenan (CAR) induces a stable hyperalgesic response (enhanced pain perception)

throughout the duration of the experiment. Treatment with compound 3 (ꢁ, 6 mg/paw) or synthetic sEH

inhibitor (TCC) (▼, 6 mg/paw) significantly reduced pain levels (Kruskal-Wallis One Way ANOVA on Ranks,

p ꢀ 0.001, Tukey’s post hoc test (compound 3 vs vehicle, TCC vs vehicle, compound 3 vs TCC, p < 0.05).

Mean ± SE (n = 6) of mechanical withdrawal threshold (% of naïve baseline) are shown. (B) Plasma

concentration of compound 3 in the pain assay after dermal treatment of compound 3 (6 mg/paw).

Concentration of demethoxy metabolite compounds 6 & 7 were below 200 nM. Blood samples were

processed by the method described for pharmacokinetic analysis. Mean ± SE (n = 6) are shown.

https://doi.org/10.1371/journal.pone.0176571.g005

= 1.3 μM, Fig 4), this metabolite could also contribute to the biological activity. Based on the

area under the curve values of compound 3 and compound 6, approximately 66% of com-

pound 3 appears to be metabolized into compound 6. On the other hand, the bis-demethoxy

compound (compound 7) was not detected (LOQ < 9.8 nM in blood). Phase II metabolism

such as glucuronidation or sulfation may be the dominant pathways for the metabolism of

compound 6.

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Plant sEH inhibitors

Fig 6. Compound 3 is orally bioavailable, and rapidly metabolized into demethoxy-metabolite compound 6. Male Swiss Webster

mice were orally treated (by gavage) with 10 mg/kg of compound 3 and 1-(adamantan-1-yl)-3-(5-(2-(2-ethoxyethoxy) ethoxy) pentyl) urea

(AEPU), and 0.3 mg/kg of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) formulated in PEG300 by cassette dosing (all

three compounds given in one cassette). Compound 3, a demethoxy-form metabolite of compound 3 (ꢁ, compound 6), AEPU and TPPU

were measured by LC-MS/MS. Bis-demethoxy compound (compound 7) was not detected (LOQ < 9.8 nM in blood). Mean ± SE (n = 4) of

blood concentrations are shown. Recently developed synthetic sEH inhibitor TPPU is much more bioavailable than many other compounds

tested and is more metabolically stable. The data show compound 3 to be of sufficient oral availability and stability to be of possible

pharmacological interest.

https://doi.org/10.1371/journal.pone.0176571.g006

Effect of incubation conditions on the occurrence of compound 1 from

papaya seed

Next, possible synthetic pathways for compound 1 was explored. We hypothesized the biosyn-

thetic pathways of benzylurea derivatives in these plants as shown in Fig 7. Conversion of glu-

cosinolates into isothiocyanates by myrosinase is a well-recognized metabolic pathway in

Brassicales [43]. Conversion of benzyl isothiocyanate into benzyl isocyanate by human P450s

has also been reported [44,45]. Although the oxidative enzyme that catalyzes this reaction in

plants has not been identified, it is possible that plants also catalyze this reaction via a mecha-

nism similar to that of human P450. Isocyanates can react with water to yield free amines or

they can react directly with amines to form 1, 3-disubstituted ureas (pathway 1). Another

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Plant sEH inhibitors

Fig 7. Proposed biosynthetic pathway of 1, 3-dibenzylurea derivatives.

https://doi.org/10.1371/journal.pone.0176571.g007

possible pathway is the conversion of isothiocyanates into thioureas by reacting with benzyla-

mines, which are then oxidized to 1, 3-dibenzylureas (pathway 2). Papaya seed was selected as

a model system to study these pathways because of the ease of obtaining fresh samples and the

relatively high concentration of benzyl isothiocyanate [46,47]. Moreover, it is incorporated

into our diet [48] and is used as an active ingredient in an anthelmintic [35].

As shown in Table 2, compound 1 was not observed in samples without overnight incuba-

tion, demonstrating that compound 1 is produced during the incubation. This is consistent

Table 2. Effect of incubation conditions and trapping experiment on concentration of compounds 1 and 4 from papaya seed.

Concentration of compound 1

(ng/g fresh seed weight)

Concentration of compound 4

(ng/g fresh seed weight)

Control

(overnight incubation)

56 ± 11

<LOQ

Without overnight incubation

Methanol treatment

<LOQ

14 ± 5

7 ± 6

-

Microwave heating

(100˚C 20 min in sealed tube)

Liquid nitrogen

<LOQ

-

4-isopropylaniline

(10 μM, 2 mL) spike

14 ± 0.5

975 ± 186

Compound 5

(10 μM, 2 mL) spike

43 ± 10

<LOQ

34 ± 9

249 ± 18

<LOQ

Compound 5

(10 μM, 2 mL) spike without papaya

1, 3-Dibenzylthiourea

(10 μM, 2 mL) spike without papaya

561 ± 108

LOQ: 1.2 ng (compound 1)/g and 2.6 ng (compound 4)/g fresh seed weight. Mean ± SD values are shown.

https://doi.org/10.1371/journal.pone.0176571.t002

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Plant sEH inhibitors

with our hypothesis that production of isothiocyanates during incubation is essential for the

production of ureas. Treatment of samples with methanol, microwave heating, or liquid nitro-

gen prevented the production of compound 1, strongly indicating the involvement of an enzy-

matic process. Compound 1 was not observed when the typical extraction procedure with

liquid nitrogen for glucosinolates was used. This observation explains why these urea deriva-

tives have been rarely detected in Brassicales.

Analysis of isocyanate, isothiocyanate, and thiourea derivatives from

papaya seed

Trapping experiments were performed in order to study the possible involvement of isocya-

nate, isothiocyanate, and thiourea in the biosynthesis of compound 1 [45]. These intermedi-

ates, especially the isocyanate, are chemically unstable and difficult to quantify. Both

isocyanate and isothiocyanate can be trapped with 4-isopropylaniline, resulting in 1-benzyl-3-

(4-isopropylphenyl) urea (compound 4) and 1-benzyl-3-(4-isopropylphenyl) thiourea (com-

pound 5), respectively. An LC-MS/MS method was optimized in order to analyze these deriva-

tives in addition to endogenous benzylurea and thiourea. Sufficient compound 4 was

recovered with the same extraction method (Recovery: 107 9%) used for the extraction of

compound 1 from papaya samples, but the concentration and recovery percentage of 1,

3-dibenzylthiourea and compound 5 varied significantly, possibly because of the instability of

the thiourea. Thus, only qualitative analysis was performed for these thiourea derivatives.

When 4-isopropylaniline was spiked into fresh papaya seeds, compound 4 was observed at

high concentration and consistent with this, the level of compound 1 decreased slightly

(Table 2). In addition, compound 5 was observed in these samples. Interestingly, compound 4

was also observed when compound 5 was spiked into papaya seed before incubation. This

strongly suggests that biosynthetic pathway 2 is at least part of the synthetic pathway of urea

derivatives. When compound 5 was incubated overnight in water at room temperature with-

out papaya seed, surprisingly, we observed high levels of compound 4. Similarly, 1, 3-diben-

zylthiourea was converted into compound 1 under these conditions, suggesting that the

oxidation of thiourea into urea could be a spontaneous non-enzymatic chemical reaction.

Approximately 10% of 1, 3-dibenzylthiourea was converted into compound 1. In addition, it

should be noted that benzyl isocyanate was not observed in the GC-MS analysis of papaya seed

(S7 Fig).

Screening of plant library in the order Brassicales on sEH inhibition and

occurrence of urea derivatives

A library of plants in the order Brassicales was constructed and screened for sEH inhibitory

activity. We mainly focused on sprouts because of the following reasons; (1) relative high

amount of glucosinolate and myrosinase activity was reported in Brassicales [49], (2) easy to

obtain fresh samples, (3) sprouts of several Brassicales are used as food.

As shown in Fig 8, screening using the fluorescent-based assay clearly prioritized the plant

extracts with regard to sEH inhibition. Plants in the Brassica genus did not show potent activ-

ity except for Redcabbage (Brassica oleracea) even though the library contained 17 species in

the genus Brassica. This tendency may be because this genus usually produces glucosinolates

with short linear aliphatic chains such as sulforaphane [49–51]. According to the previous

structure-activity relationship information, bulky group on at least one side of urea gives

higher sEH inhibitory activity than short aliphatic chains [52].

The Brassicales library was then screened for the occurrence of urea derivatives using

LC-MS/MS (S2 Table & Table 3). Compound 1 (1, 3-dibenzylurea) was found in the sprouts of

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Plant sEH inhibitors

Fig 8. Screening results of plant library in the order Brassicales against human sEH inhibitory

potency. IC₅₀was measured using cyano (2-methoxynaphthalen-6-yl) methyl trans-(3-phenyl-oxyran-2-yl)

methyl carbonate (CMNPC) as a substrate. The fluorescent-based assay as performed here has a standard

error between 10% and 20%, suggesting that differences of two-fold or greater are significant. Inhibitory

potency was expressed as -log (IC₅₀(μg plant extract/mL)/100). Dotted line represents IC₅₀= 12.6 μg/mL.

Extracts with IC₅₀above this value (below the dotted line on the graph) are considered not active. IC₅₀values

and plant names are given in S3 Table.

https://doi.org/10.1371/journal.pone.0176571.g008

Table 3. Summary of the screening of Brassicales library for the occurrence of urea derivatives.

#

Plant

Part

Human sEH IC₅₀

(μg extract/mL)

Compound 1

(nmol/g extract)

Compound 3

(or MMU, nmol/g extract)

Compound 8

(1,3-diphenetyl urea, nmol/g extract)

2

Water cress

Nasturtium

Horse radish

Garden cress

Moringa

sprout

root

1.4

51.4

34.9

33.2

23.9

21.8

5.9

129

43

3

1524

4

34

6

sprout

root

1592

6576

7

13

26

28

29

30

31

32

34

36

38

39

50

Land Cress

Moringa

sprout

root

2820

469

53

Salvadora

Limnanthes

Maca

100.0

50.0

4.5

sprout

root

7413

10

18

Maca

sprout

seed

6.3

33

Black mustard

Alyssum

31.3

14.3

50.0

13.7

35.0

13.6

25

29

45

Water cress

Curled mustard

Horse radish

Water cress

sprout

leaf

13

stem

21

Only detected urea derivatives are listed in the table. Note that the concentration is semi-quantitative, and matrix effects may interfere/enhance the

ionization.

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Plant sEH inhibitors

garden nasturtium (Tropaeolum majus) and the root of Salvadora (Salvadora persica), in addi-

tion to garden cress and Moringa (Moringa oleifera). Garden nasturtium and Salvadora have

been reported to produce benzyl glucosinolate [25,51,53]. Compound 8 (1, 3-diphenethyl

urea) was detected in the sprout of land cress (Barbarea verna) at the highest concentration,

although the extract showed only weak sEH inhibitory potency. The occurrence of compound

8 in land cress is consistent with previous reports that this plant is rich in phenethyl glucosino-

late [51,54,55]. Given the high concentration of phenethyl glucosinolate in land cress, this

plant may be ideal for further optimization of sEH inhibition. A trace amount of compound 8

was detected in sprouts of garden nasturtium, horse radish (Armoracia rusticana), and alyssum

(Lobularia maritima), consistent with the previous reports of the occurrence of phenethyl glu-

cosinolate or phenethyl isothiocyanate in these plants [56]. Methoxy benzyl urea was detected

in the sprouts of Limnanthes (Limnanthes douglasii), maca, black and curled mustard (Brassica

nigra and juncea), and the leaf of horse radish. Limnanthes has been reported to produce meta

methoxy benzyl glucosinolate, thus it is likely that compound 3 (meta methoxy benzyl urea)

was produced from this plant [51,55,57,58]. When comparing the occurrence of these urea

derivatives with sEH inhibitory potency, water cress, Moringa, and maca, which produce urea

derivatives, showed high inhibitory potency on sEH, while several other plants that produce

urea derivatives showed limited inhibitory potency.

Quantification of 1, 3-diphenethyl urea from water cress

In order to validate the screening results, we focused on water cress for the following reasons; (1)

high inhibitory potency on human sEH, (2) the ease of cultivation and availability of fresh vege-

tables, (3) reports of a high concentration of phenethyl glucosinolate [59]. Thus we focused on

diphenethyl urea compound 8, which may contribute to the observed sEH inhibitory potency of

water cress extract. First, the sEH inhibitory potency of compound 8 and related compounds

was measured (Fig 4). As expected from previous structure-activity relationships, compound 8

showed moderate sEH inhibitory potency, while phenethyl thiourea and isothiocyanate were not

potent sEH inhibitors. These data suggest that conversion of thiourea into urea could increase

the potency of extracts. Next, we looked for compound 8 in water cress. This compound was

detected and quantified in various parts of the plant (Table 4). Spike and recovery experiments

showed that our method sufficiently extracts compound 8 from the samples (Recovery:

80 5%). Leaf samples showed the highest sEH inhibitory potency. As expected, leaf samples

had the highest concentration of compound 8 when compared per extract weight. Interestingly,

seed extracts did not show inhibitory potency, although they contain a moderate concentration

of compound 8, suggesting that components other than compound 8 may be responsible for the

sEH inhibitory potency. To test this possibility, fraction collection experiments were performed

(Fig 9, S2 Text and S5 Table). The fraction containing compound 8 showed only 5% of total

inhibitory potency, while other fractions, especially lipophilic fractions showed high potency

toward sEH. Identification and characterization of these components are subjects of further

study. It should be noted that sprout extracts in these experiments showed much lower inhibi-

tory potency compared to the samples in the initial screening. This discrepancy could be due to

the different batches of the seed samples, or slight change of growth conditions.

Leaves of water cress are used as food, and several health benefits from their consumption

have been reported [60]. Oral bioavailability as well as metabolism of compound 8 needs to be

determined to relate the occurrence of compound 8, sEH inhibition, and the health benefits of

water cress. A possible hydroxylated metabolite of compound 8 showed slightly higher inhibi-

tory potency on sEH than the parent, suggesting that this compound could also contribute to

the biological activity in vivo.

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Plant sEH inhibitors

Table 4. Concentration of compound 8 in various parts of water cress.

Human soluble epoxide hydrolase IC₅₀Concentration of compound 8 Concentration of compound 8 Concentration of compound 8

(μg/g extract)

7.0 ± 1.1

17 ± 2

(μg/g extract)

(μg/g dry weight)

(μg/g fresh weight)

Leaf

Sprout

Stem

Seed

48 ± 15

2.7 ± 0.9

0.38 ± 0.09

3.2 ± 1.4

11 ± 4

0.7 ± 0.3

0.11 ± 0.04

13.6 ± 1.0

>50

0.8 ± 0.3

0.07 ± 0.03

12 ± 4

1.4 ± 0.2

Mean ± SD values are shown.

https://doi.org/10.1371/journal.pone.0176571.t004

A few papers have reported benzyl urea derivatives from plants, but their synthetic path-

ways or biological activities have not been studied extensively. Based on our data, it is likely

that conversion of thiourea into urea (Fig 7, pathway 2) is at least part of the natural synthetic

pathway of the urea derivatives reported in these earlier studies, given the processing method

they reported. To our knowledge there are no reports of spontaneous oxidation of thiourea

Fig 9. sEH inhibitory potency of HPLC fractions of water cress extract. Crude extract of water cress

sprout (approximately mg) was injected into a reverse phase HPLC column (Waters SunFire Prep C18, 5 μm,

10x100 mm) and eluted with 10% acetonitrile in water with a flow rate of 2 mL/min for 10 min, followed by a

linear gradient elution of acetonitrile 10% to 100% at a flow rate of 2 mL/min for 25 min, and eluted with 100%

acetonitrile for 15 min at a flow rate of 2 mL/min. The relative intensity of the UV absorption at the wavelength

of 210 and 280 nm are shown (top and middle). The retention time of the synthetic urea (compound 8) is

indicated by an arrow in the figure. Fractions were collected for every 4 mL of eluent. After the solvent was

evaporated the residue was reconstituted in 50 μL DMSO. The inhibition percentage by each fractions (100

times dilution of reconstituted solution) was measured using the CMNPC assay with recombinant human sEH

(bottom). The black circles (●) represent the inhibition percentage by each of the fractions. The crude extract

(2 mg extract/mL) showed complete inhibition of sEH activity.

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into urea without a special chemical reagent or catalyst. There are several synthetic methods to

convert thiourea into urea such as using 1-butyltriphenylphosphonium dichromate, potassium

monopersulfate [61], or bromine water within the pH range 2–4 [62]. Compared to these, our

method is simple and mild (neutral pH at RT), and does not require any chemical reagent

except for water and possibly oxygen from air. Determining the mechanism and substrate

specificity of the oxidation reaction should help determine whether other disubstituted ureas

occur in Brassicales. Moreover, optimization of this reaction should increase the yield of urea-

based sEH inhibitors from Brassicales.

Because the conversion of thioureas into ureas is a spontaneous chemical reaction, we pre-

dict that the occurrence of urea derivatives is conserved among Brassicales that produce gluco-

sinolate and isothiocyanate. Plants that have been reported to contain benzylurea derivatives

have diverse genetic backgrounds and are widely spread among families in Brassicales. Deter-

mining the generality of the occurrence of urea derivatives in other Brassicales might lead to the

discovery of more potent and metabolically stable urea-based sEH inhibitors from Brassicales.

In practice, urea derivatives may be generated during the preparation of traditional medi-

cines or cooking process of vegetables from plants in the Brassicales. Soluble epoxide hydrolase

inhibitors have been reported to be effective treatments in various animal disease models,

including models of inflammatory and diabetic neuropathic pain [4,5,7,12], hypertension [63],

cardiac hypertrophy [64], chronic obstructive pulmonary disease [65], and fibrosis [66]. More-

over, in combination with omega-3 fatty acid metabolite or cyclooxygenase-2 inhibition, sEH

inhibitors synergistically inhibit angiogenesis thus effectively suppress tumor growth and

metastasis [67,68]. Urea-based sEH inhibitors may contribute, at least in part, to the beneficial

effects of Brassicales, in addition to the well-known Nrf2-Keap1 pathway modulated by iso-

thiocyanates [49,69,70]. We believe that these urea derivatives are an important class of chemi-

cals that occur in nature, or more importantly, occur in our daily life.

These urea derivatives have potential for nutraceutical or pharmaceutical application for

the treatment of pain and other pathological conditions in which sEH inhibitors are effective.

From the point of view of nutraceutical or clinical application for the treatment of pain, a topi-

cal route is convenient and may be advantageous considering the rapid metabolism and high

permeability of the compound through skin.

In addition to sEH inhibitors, the 1, 3-disubstituted urea moiety is frequently used in

medicinal chemistry and is reported to possess various biological activities [71–78]. For exam-

ple, many of the kinase inhibitors, including the marketed anticancer drugs sorafenib and

regorafenib, have 1, 3-disubstituted urea-based structure with antitumor activity [79]. Urea

derivatives from Brassicales may interact with these target proteins.

Conclusions

Urea derivatives were detected in multiple plants in the order Brassicales, and their synthetic

pathways were studied, suggesting a thiourea as an intermediate. Meta methoxy substituted

dibenzylurea derivatives that possess sEH inhibitory activity were isolated and characterized

from maca root. The most potent compound (compound 3) showed the expected analgesic

efficacy on inflammatory pain and was orally available. Although urea derivatives in nature

have not been studied extensively, this class of compounds could provide unique chemical

diversity in the field of natural products and may have implications for human health.

Supporting information

S1 Text. DNA extraction and sequencing of ribosomal DNA partial sequences.

(PDF)

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Plant sEH inhibitors

S2 Text. Methods for HPLC fraction collection and sEH inhibition by the fractions.

(PDF)

S1 Table. Optimum mass transition conditions and key fragmentation of ureas and thio-

ureas.

(PDF)

S2 Table. List of urea/thiourea derivatives, mass transition conditions, and key fragmenta-

tion for MS screening of Brassicales plant library.

(PDF)

S3 Table. HPLC solvent gradients for the analysis of urea compounds.

(PDF)

S4 Table. Information of Brassicales plant library.

(PDF)

S5 Table. Relative potency of reverse phase-HPLC fractions.

(PDF)

S1 Fig. NMR spectra of compound 1 (synthetic standard & sample isolated from maca).

(PDF)

S2 Fig. NMR spectra of compound 2 (synthetic standard & sample isolated from maca).

(PDF)

S3 Fig. NMR spectra of compound 3 (synthetic standard & sample isolated from maca.

(PDF)

S4 Fig. HRESIMS spectra of compound 1 isolated from maca.

(PDF)

S5 Fig. HRESIMS spectra of compound 2 isolated from maca.

(PDF)

S6 Fig. HRESIMS spectra of compound 3 isolated from maca.

(PDF)

S7 Fig. GC-MS analysis of benzyl isocyanate and benzyl isothiocyanate.

(PDF)

S8 Fig. Intraplantar administration of compound 3 effectively reduces carrageenan-

induced inflammatory pain in rat.

(PDF)

Acknowledgments

We would like to express our sincere thanks and appreciation to Dr. E. Dean at UC Davis Cen-

ter for Plant Diversity for plant identification and preparation of plant specimens, Dr. G. R. De

Nicola at Consiglio per la Ricerca e la sperimentazione in Agricoltura, Centro di Ricerca per le

Colture Industriali (CRA-CIN) for invaluable suggestions and discussions, Dr. K. Wagner and

Dr. N. Vasylieva for fresh garden cress sprout and papaya seed samples, and Dr. A. Rand and

Dr. B. Barnych for invaluable suggestions and critiques on the manuscript.

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Plant sEH inhibitors

Author Contributions

Conceptualization: SK.

Funding acquisition: SK BDH.

Investigation: SK CM BI TRH.

Methodology: SK CM BI TRH.

Project administration: CM BDH.

Resources: BDH.

Supervision: CM BDH.

Validation: BDH.

Visualization: SK CM BI TRH BDH.

Writing – original draft: SK.

Writing – review & editing: CM BI TRH BDH.

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