Potent natural soluble epoxide hydrolase inhibitors from Pentadiplandra brazzeana Baillon: Synthesis, Quantification, and Measurement of Biological Activities In Vitro and In Vivo

Article abstract of DOI:10.1371/journal.pone.0117438

We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) i

Full text of DOI:10.1371/journal.pone.0117438

RESEARCH ARTICLE
Potent Natural Soluble Epoxide Hydrolase
Inhibitors from Pentadiplandra brazzeana
Baillon: Synthesis, Quantification, and
Measurement of Biological Activities In Vitro
and In Vivo
1
1
1
1
Seiya Kitamura , Christophe Morisseau , Bora Inceoglu , Shizuo G. Kamita ,
a11111
2
3
1
Gina R. De Nicola , Maximilienne Nyegue , Bruce D. Hammock *
1
Department of Entomology and Nematology, and University of California Davis Comprehensive Cancer
Center, University of California Davis, Davis, California, United States of America, 2 Consiglio per la Ricerca
e la sperimentazione in Agricoltura, Centro di Ricerca per le Colture Industriali (CRA-CIN), Bologna, Italy,
3
Départment of Biochemistry and Départment of Microbiology, University of Yaoundé I, Yaoundé,
Cameroon
*
bdhammock@ucdavis.edu
OPEN ACCESS
Citation: Kitamura S, Morisseau C, Inceoglu B,
Kamita SG, De Nicola GR, Nyegue M, et al. (2015)
Potent Natural Soluble Epoxide Hydrolase Inhibitors Abstract
from Pentadiplandra brazzeana Baillon: Synthesis,
Quantification, and Measurement of Biological
Activities In Vitro and In Vivo. PLoS ONE 10(2):
e0117438. doi:10.1371/journal.pone.0117438
We describe here three urea-based soluble epoxide hydrolase (sEH) inhibitors from the
root of the plant Pentadiplandra brazzeana. The concentration of these ureas in the root was
quantified by LC-MS/MS, showing that 1, 3-bis (4-methoxybenzyl) urea (MMU) is the most
abundant (42.3 μg/g dry root weight). All of the ureas were chemically synthesized, and
their inhibitory activity toward recombinant human and recombinant rat sEH was measured.
The most potent compound, MMU, showed an IC50 of 92 nM via fluorescent assay and a Ki
of 54 nM via radioactivity-based assay on human sEH. MMU effectively reduced inflamma-
tory pain in a rat nociceptive pain assay. These compounds are among the most potent sEH
inhibitors derived from natural sources. Moreover, inhibition of sEH by these compounds
may mechanistically explain some of the therapeutic effects of P. brazzeana.
Academic Editor: Monika Oberer, University of
Graz, AUSTRIA
Received: October 7, 2014
Accepted: December 23, 2014
Published: February 6, 2015
Copyright: © 2015 Kitamura et al. This is an open
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 (EpFAs) to their corresponding vicinal diols in humans and other mam-
mals [1]. These EpFAs include the epoxides of linoleic, arachidonic, eicosapentaenoic, and
docosahexaenoic acid that are produced primarily by cytochrome P450s. These natural mole-
cules 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 [2,5,6], and lower
blood pressure [7] in several in vivo models. Recently, sEH inhibitors were shown to be
Funding: This work was partially funded by National
Institute of Environmental Health Sciences (NIEHS)
grant R01ES002710, NIEHS Superfund Research
Program grant P42 ES04699, National Institute Of
Arthritis And Musculoskeletal And Skin Diseases of
the National Institutes of Health (NIH) under Award
Number R21AR062866, and NIH Counter Act
Program U54 NS079202-01. The content is solely the
responsibility of the authors and does not necessarily
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
represent the official views of the NIH. SK is
financially supported by Japan Student Services
Organization. BDH is a George and Judy Marcus
Senior Fellow of the American Asthma Foundation.
The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
effective against neuropathic diabetic pain in rodent models [8], and against equine laminitis
which is a complex and often fatal disease involving inflammation, hypertension, and severe
neuropathic pain [9]. Consequently, sEH has emerged as a potential pharmaceutical target.
One sEH inhibitor, AR9281, has undergone a phase II clinical trial for the treatment of hyper-
tension and impaired glucose tolerance [10]. Two other clinical trials are now underway with a
different sEH inhibitor that targets chronic obstructive pulmonary disease by Glaxo Smith
Kline [11,12]. Several recent studies show that at least some of the beneficial effects associated
with dietary supplementation of omega-3 fatty acids (fish oils) are due to the corresponding ep-
oxide metabolites of omega-3 fatty acids [2,13]. Thus sEH inhibitors appear to enhance the
positive effects of diet supplementation with fish oils.
A few sEH inhibitors from natural products have been identified. Buscato et al. (2013)
screened a library of compounds isolated from entomopathogenic bacteria and reported iso-
propylstilbene as an sEH inhibitor with an IC50 of 10 μM [14]. Additionally, Lee et al. (2013)
reported honokiol and β-amyrin acetate as sEH inhibitors with IC50 values of 3.4 μM and 0.57
μM, respectively [15]. However, the potency of these natural compounds is significantly lower
than that of synthetic sEH inhibitors which can possess IC50 values in the low nM to pM range
Competing Interests: The authors of this manuscript
have the following competing interests: The authors
do not have a patent relating to material pertinent to
this article. Authors BI and BDH are co-founders of
Eicosis LLC. BI, CM and BDH are co-inventors 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 of these filings
is publicly available at the USPTO website. The
numbers and titles of the patents are available on
request. No patents have been filed related to the
material in this manuscript or on any closely related
research. This does not alter the authors’ adherence
to all of the PLOS ONE policies on sharing data and
materials.
[16,17]. Although purified natural products have been shown to function as sEH inhibitors in
vitro, the in vivo efficacy of these natural products as sEH inhibitors is yet to be reported.
In our efforts to search for potent sEH inhibitors from natural products and elucidate their
possible therapeutic and nutraceutical applications, we focused on the central pharmacophore
of known sEH inhibitors with high potency. The 1,3-disubstituted urea is known as a pharma-
cophore of potent sEH inhibitors [18]. The urea pharmacophore mimics both the epoxide sub-
strate and the transition state of epoxide hydrolysis, leading to competitive inhibition of sEH.
Lipophilic substitutions on the urea are favored for improved potency [19].
Tsopmo et al. reported the isolation and identification of disubstituted urea compounds in
the root of the plant Pentadiplandra brazzeana [20]. Pentadiplandra brazzeana (commonly
known as Oubli in French) is the sole species in the plant genus Pentadiplandra. This plant
grows either as a shrub or as a liana, and is native to Cameroon and other places in West and
Central Africa [21–23]. The root of this plant is used as a folk remedy against hemorrhoids
[24], toothache [25], and as an analgesic for the treatment of chest, abdominal, and intercostal
pain, as well as rheumatic disorders [23]. Moreover, the essential oil obtained from the root has
anti-inflammatory effects [26]. Tsopmo et al. have isolated and identified three 1,3-dibenzyl
ureas in the root of P. brazzeana [20]. However, the biological activity of these ureas was
not evaluated.
On the basis of structural analogy, we hypothesized that urea compounds in P. brazzeana
are inhibitors of human sEH. To test this hypothesis we measured the inhibitory potency of the
crude root extract as well as the individual ureas found in P. brazzeana against recombinant
human and recombinant rat sEH. The amount of these inhibitors was quantified using LC-
MS/MS, and the analgesic efficacy of the most potent and abundant compound (MMU) was
measured in a nociceptive assay using a rat inflammatory pain model.
Materials and Methods
General
All reagents and solvents were purchased from commercial suppliers and were used without
further purification. Honokiol (purity>98%) was purchased from R&D systems (Minneapolis,
MN) and stored at 4°C. All of the synthetic reactions were performed in an inert atmosphere of
dry nitrogen or argon. Melting points were determined using an OptiMelt melting point appa-
1
13
ratus and are uncorrected. H and C-NMR spectra were collected using a Varian 600 MHz
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
spectrometer with chemical shifts reported relative to residual deuterated solvent peaks or tet-
ramethylsilane internal standard. Accurate masses were measured using a Micromass LCT
ESI-TOF-MS. FT-IR spectra were recorded on a Thermo Scientific NICOLET IR100 FT-IR
Spectrometer.
Ethics Statement
The plant samples were harvested under the authority of National Herbarium of Cameroon by
Mrs. Ada, a Cameroonian botanist at the National Herbarium of Cameroon. The National
Herbarium of Cameroon is the authority in charge of the promotion of research on plants.
Cameroonian researchers do not need permission to collect plant samples in Cameroon.
For the nociceptive assays, all of the studies were conducted in line with U.S. federal govern-
ment regulations and were approved by the Institutional Animal Care and Use Committee at
the University of California, Davis.
Plant material and sample preparation
th
The plant root samples were harvested on February 5 2010 at Elounden (Yaoundé, Camer-
oon) by Mrs. Ada, a botanist at the National Herbarium of Cameroon in Yaoundé. A voucher
specimen is kept at the National Herbarium of Cameroon in Yaoundé (Identication No.
6
538NM/01). Root material was freeze-dried, reduced to a fine powder and kept at -20°C until
used for the analysis.
DNA extraction and sequencing of ribosomal DNA and maturase K DNA
partial sequences
Total DNA in the root powder was extracted using a Qiagen DNeasy Plant Mini Kit (Qiagen, Va-
lencia, CA, USA) following the manufacturer’s protocol. The partial sequences of 18S ribosomal
DNA and maturase K DNA were amplified by PCR. The sequences of the PCR primers are as
0
follows: 18S ribosomal DNA, forward primer-1 (5 -GCCGCGGTAATTCCAGCTCCAA-
0
0
TAGCGTATATTT-3 ) and reverse primer-1 (5 -GAGTCCTAAAAGCAACATCCGCT-
0
0
GATCCCTG-3 ); and forward primer-2 (5 -GCAGTTAAAAAGCTCGTAGTTGGACC-
TTGGGATG-3 ) and reverse primer-2 (5 -TGAGACTAGGACGGTATCTGATCGTCTTC-
GAG-3 ). Maturase K DNA, forward primer-1 (5 -GGAGGAATTTCAAGTATATTTA-
GAGTTGGATAGAGTTCGGC-3 ) and reverse primer-1 (5 -CGCAAGAAATGCAAAGA-
AGAGGCATCTTTTACCCTG-3 ); and forward primer-2 (5 -GCAGTTAAAAAGCTCG-
TAGTTGGACCTTGGGATG-3 ) and reverse primer-2 (5 - TGAGACTAGGACGGTATCT-
GATCGTCTTCGAG-3 ). PCR amplification was performed with these primers using GoTaq
0
0
0
0
0
0
0
0
0
0
0
Green Master Mix (Promega, Madison, WI, USA) as follows: 95°C, 2 min; 35 cycles of 95°C,
3
0 sec; 62°C, 30 sec; and 72°C, 45 sec; followed by 72°C, 5 min. This PCR generated a 0.5 kbp-
long amplicon that was column-purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia,
CA, USA) following the manufacturer’s protocol. The sequences of PCR products were deter-
mined by the UC Davis College of Biological Sciences Sequencing Facility.
Extraction
The dried root powder (100 mg) was extracted with dichloromethane (DCM)-methanol
(
MeOH) (1:1) (3 x 2 ml) at room temperature (24 h per extraction). Concentration of the com-
bined percolates in vacuo yielded a yellowish crude extract (approximately 8 mg).
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
Fig 1. Synthetic schemes of urea compounds found in P. brazzeana. THF = tetrahydrofuran, DCM = dichloromethane, DIEA = diisopropylethylamine.
doi:10.1371/journal.pone.0117438.g001
Synthesis of ureas
1
,3-Dibenzylurea (BBU) was previously synthesized [27]. Fig. 1 shows the synthetic scheme for
the other 2 ureas described in this study.
-Benzyl-3-(4-methoxybenzyl) urea (BMU)
1
To a solution of benzylisocyanate (158 μl, 1.28 mmol) in THF (2 ml) was added 167 μl
(
1.28 mmol) of (4-methoxyphenyl) methanamine. After stirring for 10 min at room tempera-
ture, hexane was added and the resulting white crystals were collected (260 mg, 0.963 mmol,
1
7
5%). mp 139.6–140.2°C; H-NMR (DMSO-d , 600 MHz); δ (ppm) 7.31 (2H, dd, J = 7.5 Hz,
6 1
J2 = 5.0 Hz) 7.25–7.20 (m, 3H), 7.17 (2H, d, J = 8.4 Hz), 6.87 (2H, d, J = 8.4 Hz), 6.38 (1H, t, J =
6
.0 Hz), 6.33 (1H, t, J = 6.0 Hz), 4.22 (2H, d, J = 6.0 Hz), 4.15 (2H, d, J = 6.0 Hz), 3.73 (3H, s),
1
3
C NMR (151 MHz, DMSO-d ): δ 158.1, 158.0, 140.9, 132.8, 128.3, 128.2, 127.0, 126.5, 113.6,
6
-
1
5
+
5.0, 42.9, 42.4; IR (cm ) 3349, 3317, 3032, 2923, 1625, 1577, 1511, 1242, 1031, ESI-MS [M
+
Na] m/z 293.11 (calcd for C H N NaO 293.13).
16 18 2 2
1
, 3-Bis (4-methoxybenzyl) urea (MMU)
To an ice-cold solution of triphosgene (100 mg, 0.34 mmol) in 2 ml DCM was added drop-
wise a solution of 4-methoxybenzylamine (137 mg, 1 mmol) and N,N,-diisopropylethylamine
DIEA) (191 μl, 1.1 mmol) in 2 ml of DCM. This mixture was stirred for 5 min at 0°C. To this
mixture was added 4-methoxybenzylamine (137 mg, 1 mmol) and DIEA (191 μl, 1.1 mmol) in
ml of DCM, warmed slowly to room temperature, and stirred overnight. To this solution was
(
2
added DCM, and washed with 1 M aqueous HCl solution three times. The DCM layer was
dried over MgSO and concentrated. Recrystallization from the DCM resulted in 45 mg
4
1
(
0.15 mmol, 15%) of target compound as a white powder; mp 177.7–178.4°C; H-NMR
(
6
1
DMSO-d , 600 MHz); δ (ppm) 7.16 (4H, d, J = 9.0 Hz), 6.87 (4H, d, J = 8.4), 6.27 (2H, t, J =
6
13
.0), 4.14 (4H, d, J = 6.0), 3.72 (6H, s), C NMR (151 MHz, DMSO-d ): δ 158.0, 158.0, 132.8,
6
-
1
28.3, 113.6, 55.0, 42.4; IR (cm ) 3350, 3318, 2955, 2924, 2837, 1624, 1577, 1509, 1237, 808,
+
ESI-MS [M+Na] m/z 323.13 (calcd for C H N NaO 323.14).
17
20
2
3
LC-MS/MS analysis
Urea compounds were quantified 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
voltage. The source and the desolvation temperatures were set at 120 and 300°C, respectively.
Cone gas (N ) and desolvation gas (N ) were maintained at flow rates of 10 and 700 l/h, respec-
2
2
tively. Optimized conditions for mass spectrometry are shown in Table 1. Dwell time was set to
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
Table 1. Optimum mass transition conditions and key fragmentation of the urea compounds.
Ionization mode
Transition
Cone voltage (V)
Collision voltage (V)
MMU
BMU
BBU
DPU
+
+
+
+
301.2
271.3
241.2
213.1
->
->
->
->
120.9
120.9
90.9
35
33
30
35
26
23
20
16
93.9
doi:10.1371/journal.pone.0117438.t001
0
.1 s. A regression curve for each compound was obtained from at least eight different concen-
2
trations of standard stock solutions (r > 0.99). 1, 3-Diphenylurea (DPU) was used as an inter-
nal standard and was added just before the analysis. The final concentration of 1, 3-
diphenylurea was adjusted to 100 nM. Full scan MS/MS spectra (m/z interval of 10–300) were
collected for each of the molecules (precursor ion; MMU m/z: 301, BMU m/z: 271, BBU m/z:
2
41) using product ion scan mode with cone voltage and collision voltage of 35V and 26V, re-
spectively. The MS was coupled with a Waters Acquity UPLC (Waters, and Milford, MA,
USA). The HPLC column Imtakt Cadenza CD-C18 (CD007, 3 μm, 4.6x500 mm) was used to
separate these analytes. The HPLC solvent gradient is shown in Table 2. The infusion flow rate
to the MS was adjusted to 0.3 ml/min.
Standard addition and recovery experiments
A solution of BBU (20 nmol), BMU (100 nmol), and MMU (200 nmol) in 10 μl of methanol
was spiked into 100 mg of dried root powder. The powder was mixed, dried, and kept at -20°C
until extraction. Samples were extracted as described above. The recovery percentage was cal-
culated as follows:
%
recovery ¼ðamount in spiked sample ꢀ amount in controlÞ ꢁ 100 = amount spiked
Enzyme Purification
Recombinant human and recombinant rat sEH were produced in insect High Five cells using
recombinant baculovirus expression vectors, and purified by affinity chromatography as re-
ported previously [28,29]. Each enzyme appeared as a single band (0.3 μg loading) with an
Table 2. HPLC solvent gradient for the separation of target ureas.
a
b
c
Time
.00
Aqueous phase
Organic phase
0
50
32
0
50
2
2
3
3
4
5.00
5.01
5.00
5.01
0.00
68
100
100
50
0
50
50
50
a
b
c
0
.6 ml/min flow rate,
Milli-Q Water 99.9, Acetic acid 0.1, volume %,
Acetonitrile 99.9, Acetic acid 0.1, volume %.
doi:10.1371/journal.pone.0117438.t002
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sEH Inhibitors from P. brazzeana
estimated purity of more than 95% by Coomassie Brilliant Blue staining following SDS-PAGE
separation (S1 Fig.). The final recombinant sEH preparations had no esterase or glutathione
S-transferase activity which interferes with the CMNPC assay as described below.
Measurement of sEH inhibition by fluorescent assay (CMNPC assay)
IC50 values 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 human or recombinant rat sEH (0.96 nM) was incubated with crude root
extract or 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 determin-
ing the appearance of the 6-methoxy-2-naphthaldehyde with an excitation wavelength of
3
30 nm and an emission wavelength of 465 nm for 10 min.
Measurement of sEH inhibition by radioactivity-based assay (t-DPPO
assay)
IC50 values were determined as described previously [31] with slight modifications, using race-
3
mic [ H] trans-diphenylpropene oxide (t-DPPO). Purified recombinant human sEH was dilut-
ed in 100 mM sodium phosphate buffer (pH 7.4) containing 0.1 mg/ml BSA, and was
incubated in triplicate with inhibitors for 5 min at 30°C prior to the introduction of the radiola-
beled substrate (t-DPPO: 50 μM; *10,000 cpm/assay). The mixture was incubated at 30°C for
1
0 min and the reaction was quenched by the addition of 60 μl of methanol. The remaining
substrate was extracted by vigorous mixing with 200 μl of isooctane. The radioactivity of the
aqueous phase was measured using a liquid scintillation counter (Perkin Elmer Tri-Carb
2
810TR, Shelton, CT). Epoxide hydrolase activity was determined as a percentage of the radio-
activity corresponding to the diol in the aqueous phase relative to the control.
Enzyme kinetics
Dissociation constants were determined following the method described by Dixon [32] for
competitive tight binding inhibitors, using t-DPPO as a substrate [31]. Inhibitor MMU at con-
centrations between 0 and 1000 nM was incubated in triplicate for 5 min in 100 mM sodium
phosphate buffer (pH 7.4) at 30°C with 100 μl of the enzyme (2 nM of recombinant human
sEH). Substrate (4.0 ꢂ [S]
ꢂ 30 μM) was then added. Velocity was measured as described
final
previously [31]. For each substrate concentration, the plots of the velocity as a function of the
inhibitor concentration allow the determination of an apparent inhibition constant (Kiapp
[32]). The plot of the Kiapp values as a function of the substrate concentration allows the deter-
mination of Ki when [S] = 0. Kiapp was measured in at least 3 separate assays. Results are pre-
sented as average ± SE.
Measurement of solubility of MMU
An excess amount of MMU was added to a vial containing sodium phosphate buffer, 0.1 M
pH 7.4 (0.25 mL), and the MMU suspension was equilibrated during 1 h of sonication and 48 h
of shaking at 25°C, followed by centrifugation. The amount of MMU in the supernatant was
analyzed by LC—MS/MS. The result is presented as mean ± SD of triplicate measurement.
Nociceptive assay using rat inflammatory Pain Model
Male Sprague-Dawley rats weighing 235–280 g were obtained from Charles River Laboratories
and maintained in the UC Davis animal housing facility with ad libitum water and food on a
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sEH Inhibitors from P. brazzeana
1
2 h/12 h light-dark cycle. Behavioral nociceptive testing was conducted by assessing mechani-
cal withdrawal threshold using an electronic von Frey anesthesiometer apparatus (IITC,
Woodland Hills, CA) [33]. The controller was set to “maximum holding” mode so that the
highest applied force (in grams) upon withdrawal of the paw was displayed. At least three mea-
surements were taken at 1–2 min inter-stimulus intervals. Data were normalized to percentage
values using the formula (mechanical withdrawal threshold (g)) ×100/ (mechanical withdrawal
threshold (g) of naïve animals). The analgesic effect of MMU was tested using the intraplantar
carrageenan elicited local inflammatory pain model [16,33]. Following baseline measurements,
carrageenan (50 μl, 1% solution of carrageenan) was administered into the plantar area of one
hind paw in order to induce inflammatory pain. At 4 h post administration of carrageenan,
postcarrageenan responses were measured, and immediately afterwards, MMU (10 μg in 10 μl
of PEG400), morphine sulfate (10 μg in 10 μl saline), the potent synthetic sEH inhibitor 1-(1-
(
Cyclopropanecarbonyl) piperidin-4-yl)-3-(4-(trifluoromethoxy) phenyl) urea (TPCU) (10 ng
in 10 μl of PEG400), or the vehicle (10 μl of PEG400) was administered into the inflamed paw
by intraplantar injection. Subsequently, the ability of these treatments to reduce the carrageen-
an-induced inflammatory pain was monitored over the course of 2.5 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 signicant.
Results and Discussion
Inhibition of human soluble epoxide hydrolase by crude root extract
Determination of the plant species of the root tissues was supported by comparing the partial
sequences of 18S ribosomal DNA (NCBI accession number: AF070972) and maturase K DNA
(
AY483239) as described in a previous study [34]. The sequence alignments are shown in S1
Text. The crude root extract of P. brazzeana was prepared by percolation in DCM-methanol
1:1), and the inhibitory activity of the extract toward human sEH was measured by both fluo-
(
rescent assay (CMNPC assay) and radioactivity based assay (t-DPPO assay) [30,31]. The root
extract showed an IC50 value of 1.9 ± 0.4 μg crude extract/ml in the CMNPC assay and 35 ± 3
μg crude extract/ml in the t-DPPO assay.
Optimization of LC-MS/MS and analysis of plant extracts
Two of 3 urea derivatives found in P. brazzeana as reported by Tsopmo et al. [20] were syn-
thetically prepared as shown in Fig. 1. Fig. 2A shows a representative chromatogram of the sep-
aration of synthetic standards of the 3 ureas reported by Tsopmo et al. with our internal
standard, 1,3-diphenylurea (DPU). The LC-MS/MS analysis clearly showed that the plant root
extract contains these ureas, based on the retention time of the analytes and the mass to charge
ratio of the molecular and fragment ions (Figs. 2B and S2–S4).
To determine the concentration of these ureas in plant samples an LC-MS/MS method was
optimized in multiple reaction monitoring (MRM) mode. The concentrations of these ureas
are shown in Table 3. 1, 3-bis (4-Methoxybenzyl) urea (MMU) was the most abundant, fol-
lowed by an asymmetric urea (BMU) and then non-substituted benzyl urea (BBU). In order to
determine the extraction efficiency from the plant sample, standard addition and recovery ex-
periments were performed. As shown in Table 3, the recovery percentages of MMU, BMU, and
BBU were between 89–99%, showing that our method efficiently extracted the target analytes.
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sEH Inhibitors from P. brazzeana
Fig 2. Root of P. brazzeana contains urea-based sEH inhibitors. The chemical structure of target analytes and their mass transition are shown as inserts
in the figure. The LC-MS/MS conditions are described in Materials and Methods. (A) MRM chromatograms of synthetic standard ureas. (B) MRM
chromatograms of crude root extract from P. brazzeana (250 μg root/ml, 10μl). cps: counts per second.
doi:10.1371/journal.pone.0117438.g002
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sEH Inhibitors from P. brazzeana
Table 3. Concentration of ureas in the root of P. brazzeana and extraction recovery percentage.
Concentration
μg/ g dry root weight)
Recovery
(%)
1
2
(
MMU
BMU
BBU
42.3±4.2
13.7±1.5
1.9±0.2
94±2.8
89±2.9
99±5.4
1
n = 4, mean ± standard deviation are shown.
n = 5, mean ± standard deviation are shown.
2
doi:10.1371/journal.pone.0117438.t003
sEH inhibitory activity of ureas from P. brazzeana
The ability of MMU, BMU, and BBU to inhibit the activity of human sEH was measured using
two different substrates. The IC50 values of these urea compounds are shown in Table 4. As re-
ported previously, the t-DPPO assay gives higher IC50 values (lower potency) for the same
compound than the CMNPC assay [30]. The symmetric bis (methoxybenzyl) urea (MMU),
which was found at the highest concentration in the root extract, showed the highest potency
toward human sEH when determined with the CMNPC or t-DPPO assays. The inhibitory ac-
tivity of the asymmetric urea (BMU) was of about 4-fold lower than that of MMU with both as-
says. The non-substituted benzyl urea, BBU, showed the lowest potency. These results are
consistent with the previous structure-activity relationship of sEH inhibitors in that para meth-
oxy substitution on the phenyl ring leads to higher potency in comparison to non-substituted
inhibitors [16].
In order to compare our compounds with previously reported natural product sEH inhibi-
tors, the potency of honokiol was measured using our radioactivity-based assay. Honokiol
showed an IC50 of 20.3 μM with the t-DPPO assay. Based on this result, MMU is about 8-fold
more potent than honokiol.
The inhibitory potency of these compounds against affinity purified, recombinant rat sEH
was measured using the CMNPC assay. The ureas from P. brazzeana showed a similar pattern
of inhibitory potency with the rat recombinant sEH as was found with the recombinant human
sEH (Table 4).
Enzyme inhibition kinetics
In order to define the mode of enzyme inhibition and to determine the dissociation constant
(Ki), we performed enzyme kinetic analysis of MMU using recombinant human sEH. As
Table 4. sEH inhibition potency of ureas found in the root of P. brazzeana.
Human
Human
Rat
sEH IC50 (nM)
sEH IC50 (μM)
(t-DPPO assay)
sEH IC50 (nM)
(CMNPC assay)
1
1
1
(CMNPC assay)
MMU
BMU
92±14
2.6±0.4
35±5
400±50
1900±300
10.2±2.6
48.3±7.0
20.3±1.7
44±4.2
100±6
BBU
2
2
honokiol
ND
ND
1
Measured three times and mean ± standard deviation are shown.
2
ND: Not determined.
doi:10.1371/journal.pone.0117438.t004
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sEH Inhibitors from P. brazzeana
Fig 3. Enzyme inhibition kinetics of MMU on human soluble epoxide hydrolase. Enzyme kinetic
3
analysis was performed on recombinant human soluble epoxide hydrolase (2 nM) using [ H] t-DPPO as a
substrate. For each substrate concentration (4.0–30.0 μM), the velocity was plotted as a function of inhibitor
concentration (0–1000 nM), allowing the determination of an apparent inhibition constant (Kiapp). Kiapp
values are plotted as a function of the substrate concentration (insert). For [S] = 0, a Ki value of 54 nM
was found.
doi:10.1371/journal.pone.0117438.g003
shown in the insert in Fig. 3, the Kiapp values increase with an increase in the concentration of
the substrate. This strongly suggests that MMU inhibits sEH as a competitive inhibitor, which
is consistent with other urea-based sEH inhibitors [18]. The dissociation constant (Ki) of
MMU was determined to be 54±13 nM. In terms of potency this is approximately 40-fold
lower than the highly potent synthetic inhibitor t-AUCB (Ki = 1.5 nM), but approximately 2-
fold higher than the synthetic sEH inhibitor APAU (Ki = 125 nM) which has undergone a
phase II clinical trial [35,36].
Nociceptive assay in rat inflammatory pain model
Several studies have shown that sEH inhibitors effectively reduce inflammatory pain [4,5,16].
In order to evaluate the in vivo efficacy of MMU, a rat inflammatory pain model was used to
measure its analgesic effects. MMU was selected based on its inhibitory potency on sEH and its
concentration in P. brazzeana. As shown in Fig. 4, local administration of either MMU (10 μg/
intraplantar injection), morphine (10 μg/intraplantar injection), or potent synthetic sEH inhib-
itor TPCU (10 ng/intraplantar injection) significantly reduced carrageenan induced inflamma-
tory pain (Kruskal-Wallis One Way ANOVA on Ranks, pꢂ0.001). In this assay, MMU, TPCU,
and morphine appeared to have similar efficacy in terms of reducing enhanced pain perception
but MMU showed a longer duration of efficacy compared to morphine (MMU vs morphine,
Tukey’s post hoc test, p<0.05). TPCU showed an IC50 of 0.4 nM with Ki value of 0.67 nM on
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
Fig 4. MMU effectively reduces carrageenan-induced inflammatory pain in rat. Administration of the
inflammatory agent carrageenan (CAR) induces a stable hyperalgesic response for the duration of the
experiment. Treatment with MMU (▲, 10 μg/paw, intraplantar), morphine (●, 10 μg/paw, intraplantar) or the
potent synthetic sEH inhibitor (TPCU) (4, 10 ng/paw, intraplantar) significantly reduced pain levels (Kruskal-
Wallis One Way ANOVA on Ranks, pꢂ0.001, Tukey’s post hoc test (MMU vs vehicle, morphine vs vehicle,
TPCU vs vehicle, and MMU vs morphine) p<0.05). Mean ± SE (n = 6) of mechanical withdrawal threshold (%
of naïve baseline) are shown. Vehicle (ꢃ), morphine (●), and TPCU (4) treatment data are from Rose et al.,
(
2010).
doi:10.1371/journal.pone.0117438.g004
human sEH [16,36], which is approximately 230-fold more potent than MMU based on the
IC50 values. Thus, it is reasonable that MMU showed similar or slightly better efficacy in com-
parison to TPCU when MMU was administered at a 1000-fold higher dose than TPCU.
The root of P. brazzeana is used as a traditional analgesic against various types of pain
[23,25]. For example, dried root bark powder is applied by scarification to treat intercostal and
abdominal pain, while the crushed root or root bark is applied as a topical ointment or as an in-
fusion drink to soothe chest pain, lumbago, rheumatism, and haemorrhoids [23]. In order to
relate our results to the ethnomedicinal use of P. brazzeana, it is necessary to determine how
much of the ureas are absorbed into the body, and whether they reach a dose that can effective-
ly inhibit sEH. In the case of application by scarification, which is similar to intraplantar injec-
tion, these urea compounds could be absorbed and reach the site of action. In the case of oral
administration, MMU may have limited bioavailability given that the synthetic MMU has low
solubility (10.6±2.1 μM (mean±SD)) in sodium phosphate buffer (pH7.4). It should be noted,
however, that the root of P. brazzeana may have compounds such as lipids and polysaccha-
rides, which could solubilize MMU, BMU or BBU and facilitate their absorption after inges-
tion. Determination of the bioavailability of these inhibitors after oral administration of the
root, or topical ointment treatment is the subject of further research. A recent study showed
that sEH inhibitors are highly effective against neuropathic diabetic pain [8]. Determining the
efficacy of the compound/plant extract on neuropathic pain is also the subject of further study.
Determining the localization of MMU within the plant root, especially whether it is localized
within the root bark, will help us to optimize a method for efficient isolation of MMU or other
target ureas from P. brazzeana.
The theoretical IC50 of the crude extract of P. brazzeana against recombinant human sEH is
approximately 29 μg crude root extract/ml using the CMNPC assay. This calculated IC50 of the
crude root extract is based on the IC50 of MMU and its concentration in the plant (assuming
for simplicity that MMU is the main component that inhibits sEH). Experimentally, however,
PLOS ONE | DOI:10.1371/journal.pone.0117438 February 6, 2015
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sEH Inhibitors from P. brazzeana
the crude root extract gave an IC50 of 1.9 μg crude root extract/ml. This experimentally-derived
potency is approximately 15-fold higher than the calculated potency, suggesting that the crude
root extract contains unknown sEH inhibitor(s), or another factor that improves its potency. It
should be noted, however, that in different preparations of the crude root extract, significant
variation in the inhibitory potency (IC50 = 4.1–176 μg/ml in t-DPPO assay) was found while
the concentrations of MMU, BMU, and BBU were very similar. This may indicate that the
root extract contains unknown inhibitors that are sensitive to the extraction process that is uti-
lized. Optimization of the extraction/processing method is still needed in order to precisely
measure the level of sEH inhibition by the crude plant extract. In our preliminary HPLC sepa-
ration of the crude root extract and measurement of sEH inhibition by each fraction, we ob-
served strong sEH inhibition in fractions that were different from the fractions containing
MMU (S5 and S7 Figs., S1 and S2 Tables). This indicates that there are other unidentified sEH
inhibitors in this plant. Addition of dithiothreitol (100 μM) to either the crude extract or to
each fraction did not change the inhibitory potency, indicating that the unknown inhibitors
are not Michael acceptors. The purification and identification of these inhibitors are the sub-
jects of further study.
MMU and BMU have not been reported in other plants, while BBU has been detected in
plants such as moringa (Moringa oleifera) and garden cress (Lepidium sativum) [37,38]. A
regioisomer of MMU, bis (m-methoxybenzyl) urea (salvadourea), was reported in the miswak,
Salvadora persica, plant [39]. Interestingly, P. brazzeana, moringa, garden cress, and miswak
all belong to the plant order Brassicales. In addition these plants produce glucosinolates or
their corresponding isothiocyanate products [40–43], suggesting that they may share a meta-
bolic pathway. A genetic comparison between these species based on ribosomal DNA se-
quences, however, failed to find any similarity between these species. Chemotaxonomical
analysis is needed to find shared biosynthetic pathways in these plants.
Conclusions
We demonstrate that urea compounds found in the root of P. brazzeana have potent sEH in-
hibitory activities. These inhibitors were quantified by LC-MS/MS, showing that the most po-
tent inhibitor, MMU, is found at the highest concentration in the plant root in comparison to
BMU and BBU. MMU showed local analgesic effects in an in vivo inflammatory pain model.
These findings may explain, at least in part, the pharmacological mechanisms of traditional
medicinal uses of the root of P. brazzeana. Our findings illustrate that plants could provide
leads for novel and therapeutically valuable sEH inhibitors and that plant natural products that
inhibit sEH could be valuable supplements to an omega-3 fatty acid-rich diet.
Supporting Information
S1 Fig. SDS-PAGE analysis of human and rat recombinant sEH for purity assessment. Each
enzyme appeared as a single band (0.3 μg loading) of ca. 62 kDa by Coomassie Brilliant Blue
staining following SDS-PAGE separation. The migration of molecular weight markers (in kDa)
are indicated to the left.
(
TIF)
S2 Fig. Full scan MS/MS spectra of MMU synthetic standard (upper panel) and plant ex-
tract (lower panel). Full scan MS/MS spectra (m/z interval of 10–300) were collected for
MMU setting precursor ion m/z as 301 with cone voltage and collision voltage of 35V and 26V,
respectively. cps: counts per second.
(
TIF)
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sEH Inhibitors from P. brazzeana
S3 Fig. Full scan MS/MS spectra of BMU synthetic standard (upper panel) and plant extract
(
lower panel). Full scan MS/MS spectra (m/z interval of 10–300) were collected for BMU set-
ting precursor ion m/z as 271 with cone voltage and collision voltage of 35V and 26V, respec-
tively. cps: counts per second.
(
TIF)
S4 Fig. Full scan MS/MS spectra of BBU synthetic standard (upper panel) and plant extract
lower panel). Full scan MS/MS spectra (m/z interval of 10–300) were collected for BBU set-
(
ting precursor ion m/z as 241 with cone voltage and collision voltage of 35V and 26V, respec-
tively. cps: counts per second.
(
TIF)
S5 Fig. Normal phase HPLC fraction collection and sEH inhibition by the fractions. Crude
root extract (approximately 2 mg) was injected into a normal phase HPLC column (YMC-
Pack SIL-06) and eluted with 20% isopropanol in hexane with a flow rate of 4 ml/min for 15
min, followed by 100% isopropanol with a flow rate of 2 ml/min for 25 min. The relative inten-
sity of the UV absorption at the wavelength of 210 and 276 nm are shown (top and middle).
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 dilu-
tion 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 mixture (100 times dilution of 2 mg extract/ml DMSO) showed
complete inhibition of sEH activity (black circle shown at time 0 min). The retention times of
the 3 synthetic ureas (BBU, BMU, and MMU) are indicated by arrows in the figure.
(
TIF)
S6 Fig. Procedure for the reverse phase HPLC fraction collection.
TIF)
(
S7 Fig. Reverse phase HPLC fraction collection and sEH inhibition by the fractions. Crude
root extract (approximately 5 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 times of the 3 synthetic ureas (BBU, BMU, and MMU)
are 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 per-
centage 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 in-
hibition percentage by each of the fractions. The crude extract C (100 times dilution of 5 mg ex-
tract/ml DMSO) showed complete inhibition of sEH activity (black circle shown at time 0 min).
(
TIF)
S1 Table. Relative potency of normal phase HPLC fractions.
DOCX)
S2 Table. Relative potency of reverse phase HPLC fractions.
DOCX)
S3 Table. Effect of extraction solvent on human sEH inhibitory potency.
DOCX)
(
(
(
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sEH Inhibitors from P. brazzeana
S1 Text. Sequence alignments of PCR products from root sample of P. brazzeana vs se-
quences in NCBI database.
(
DOCX)
S2 Text. Methods for HPLC fraction collection and sEH inhibition by the fractions.
DOCX)
(
Author Contributions
Conceived and designed the experiments: SK. Performed the experiments: SK CM BI SGK
GRDN. Analyzed the data: SK CM. Contributed reagents/materials/analysis tools: GRDN MN.
Wrote the paper: SK CM BI SGK GRDN MN BDH. Wrote the first draft and finalized the
manuscript: SK.
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