Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation

Article abstract of DOI:10.1021/acs.jafc.0c02641

The rhizomes of Zingiber purpureum, Bangle , were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70percent of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.

Full text of DOI:10.1021/acs.jafc.0c02641

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Bioactive Constituents, Metabolites, and Functions
Metabolite profiling of Javanese ginger Zingiber purpureum
and identification of antiseizure metabolites via a low-
cost open-source zebrafish bioassay-guided isolation
Théo Brillatz, Miwa Kubo, Shimon Takahashi, Natsumi Jozukuri, Kenshi Takechi,
Emerson Ferreira Queiroz, Laurence Marcourt, Pierre-Marie Allard, Richard Fish, Kenichi Harada,
Keisuke Ishizawa, Alexander D Crawford, Yoshiyasu Fukuyama, and Jean-Luc Wolfender
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c02641 • Publication Date (Web): 06 Jul 2020
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Metabolite profiling of Javanese ginger Zingiber purpureum and
identification of antiseizure metabolites via a low-cost open-source zebrafish
bioassay-guided isolation
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Théo Brillatz,a,b# Miwa Kubo,c,#,* Shimon Takahashi,d,e Natsumi Jozukuri,e Kenshi Takechi,f
Emerson Ferreira Queiroz,a,b Laurence Marcourt,a,b Pierre-Marie Allard,a,b Richard Fish,g
Kenichi Harada,c Keisuke Ishizawa,d,e Alexander D. Crawford,h Yoshiyasu Fukuyama,c,¥ Jean-
Luc Wolfendera,b¥,*
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a School of Pharmaceutical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, University of
Geneva, CMU - Rue Michel-Servet 1, CH-1211 Geneva 4, Switzerland
b Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet
1, CH-1211 Geneva 4, Switzerland
c Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
d Department of Pharmacy, Tokushima University Hospital, Tokushima, Japan
e Department of Clinical Pharmacology and Therapeutics, Tokushima University Graduate School of Biomedical
Sciences, Tokushima, Japan
f Clinical Trial Center for Developmental Therapeutics, Tokushima University Hospital, Tokushima, Japan
g Department of Genetic Medicine and Development, University of Geneva, Faculty of Medicine, CMU - Rue
Michel-Servet 1, CH-1211 Geneva 4, Switzerland
h Department of Preclinical Sciences & Pathology, Norwegian University of Life Sciences, Ullevålsveien 72, 0454
Oslo, Norway
# These authors contributed equally to this work.
¥ These authors contributed equally to this work.
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* Corresponding authors at: Jean-Luc Wolfender: School of Pharmaceutical Sciences, Institute of Pharmaceutical
Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, CH-1211 Geneva 4,
Switzerland. Tel.: +41 223 793 385; Fax: +41 223 793 399. E-mail address: Jean-Luc.Wolfender@unige.ch (J-L.
Wolfender). Miwa Kubo: Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan.
Tel.: +81 886 028 436; Fax: +81 886 553 051. E-mail address: miwa-k@ph.bunri-u.ac.jp (M. Kubo).
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Abstract
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The rhizomes of Zingiber purpureum, “Bangle”, were investigated for its antiseizure properties
using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay.
Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was,
therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were
isolated and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to
70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate
activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity
of Z. purpureum active compounds, its chemical profile was compared to the one of Z.
officinale. Their composition was assessed by differential metabolite profiling visualized by
molecular network, which revealed only vanillin derivatives and terpenoids as common
metabolites and gave a comprehensive view of Z. purpureum composition. This study
demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable
for routine screening in phytochemistry laboratories.
Keywords: Zingiber purpureum Rosc.; metabolite profiling; epilepsy; zebrafish; bioactivity-
guided isolation
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Epilepsy is a chronic neurological disorder affecting more than 50 million people worldwide.1-2
Despite the availability of more than 30 antiseizure drugs (ASDs), one-third of people with
epilepsy are drug-resistant, which significantly reduces their quality of life.3 There is therefore
an urgent need for cost-effective ASDs to treat epilepsy and, in this regard, plant-based
remedies could be a significant source for new ASD drug leads.4
The rhizomes of Zingiber purpureum Rosc. (synonyms of Z. cassumunar Roxb. and Z.
montanum (Koenig) Link ex A.Dietr.), also called “Bangle”, is a tropical ginger widely
distributed in Southeast Asia.5 Used as a spice, this plant finds its application in the traditional
Indonesian medicine Jamu and has been used to treat fever, headache, stomach pain,
rheumatism, obesity, and as an ingredient in postpartum herbal medicine.6 In our previous
studies, trans- (11) and cis-banglene (12) were successfully isolated from the methanolic
extract of Z. purpureum rhizomes and showed neurotrophic activity in PC12 cells.7-9
A
synthetic route was devised for 11 and 128 and, in addition, a phenylbutenoid derivative was
isolated and showed inhibition of Aβ42 aggregation.9 Furthermore, chronic treatments with 11
and 12 were found to enhance hippocampal neurogenesis in a dementia model of olfactory
bulbectomized (OBX) mice.8 These results suggested that Z. purpureum could be developed
as a valuable food additive with potential properties against neurodegenerative diseases.10 In
this context, and as Z. purpureum possesses central nervous system (CNS)-targeting properties,
our investigation focuses on its potential antiseizure activity in the present study.
Natural products have a strong potential to treat epilepsy11 and this has been demonstrated by
the recent approval of cannabidiol (CBD), the first antiseizure drug of natural origin used to
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treat rare forms of epilepsies.12 To date, a range of in vitro and in vivo models have been
developed to investigate the antiseizure potential of natural products.13 In the present study, a
zebrafish epilepsy model was chosen as a screening method in order to find novel antiseizure
drug leads. This model relies on the chemical induction of seizures by the GABAA antagonist
pentylenetetrazole (PTZ) and its effects are quantified by behavioral and/or
electrophysiological analyses.14 The zebrafish model has a number of advantages for high-
throughput drug discovery screening at the microgram scale.15 To date, this zebrafish epilepsy
model efficiently identified antiseizure compounds from natural extracts such as the nucleoside
inosine from the diatom Skeletonema marinoi,16 spirostane glycosides from Solanum torvum,17
bisabolene sesquiterpenoids from Curcuma longa,18 and sesquiterpenoids from Cyperus
articulatus.19 However, this in vivo model requires, in general, costly instrumentations for
locomotor activity screening such as the Zebrabox™ (Viewpoint, Lyon, France) or
DanioVision™ (Noldus Information Technology, Wageningen, Netherlands). In contrast,
open-source software are well-developed and provide a cost-effective solution for simple and
easy behavioral phenotype measurement.20-22 So far, no zebrafish platforms based on
affordable equipment and open-source software have been used for the bioactivity-guided
isolation of antiseizure compounds.
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The present study describes the first adaptation of a streamlined zebrafish epilepsy assay based
on low-cost equipment and open-source automated tracking software20 for bioactivity-guided
isolation. This assay was used for the identification of the active principles of Z. purpureum
prior to their evaluation in rodents. Finally, a differential metabolite profiling was performed
to study the specificity of Z. purpureum metabolites compared to those of the common Zingiber
officinale.
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Reagents. Pentylenetetrazole (PTZ) was purchased from Sigma (Sigma-Aldrich, St. Louis,
MO, USA).
General experimental procedures. NMR spectroscopic data were recorded on a Bruker
Avance III HD 600 MHz NMR spectrometer equipped with a QCI 5 mm Cryoprobe and a
SampleJet automated sample changer (Bruker BioSpin, Rheinstetten, Germany) or on a Bruker
Avance III HD 500 MHz NMR spectrometer equipped with a BBO 5 mm Cryoprobe. Chemical
shifts (δ) are measured in parts per million (ppm) using the CDCl3 signal as internal standard
for all spectra (δH 7.27; δC 77.2) and coupling constants (J) are reported in Hz. The complete
assignment was performed based on two-dimensional experiments (COSY, NOESY, HSQC,
HMBC, NOESY, and ROESY). High-resolution tandem mass spectrometry (HRMS/MS) data
were obtained on a Q Exactive Focus quadrupole-orbitrap mass spectrometer (Thermo
Scientific, Bremen, Germany) using heated electrospray ionization (HESI-II) in the positive
mode. Reverse phase analysis was performed on high-performance liquid chromatography
(HPLC) Agilent 1260 Infinity II LC series system consisting of a degasser, a mixing pump, an
autosampler, and a diode array detector (DAD) (Agilent Technologies, Santa Clara, USA),
connected to an evaporative light scattering detector (ELSD) Sedex LT-ELSD 85 (Sedere,
Alfortville, France) to detect non-UV absorbing components. Fractionation was performed on
a semi-preparative HPLC equipment (Armen modular spot prep II, Saint-Avé, France).
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Plant material. Rhizomes of Zingiber purpureum Rosc. were purchased from an Indonesian
market by Hosoda Co. Ltd. in April 2013 and identified by Dr. E. Kato (Hosoda Co. Ltd.
company). A voucher specimen (n°1799) has been deposited in the Institute of Pharmacognosy,
Tokushima Bunri University, Japan. Rhizomes of Zingiber officinale Rosc. were purchased
from Dixa Co. Ltd. (Dixa AG, Switzerland).
Preparation of extracts. The rhizome powder of Z. purpureum (10.1 g) was successively
extracted by solvents of increasing polarity (hexane, ethyl acetate, methanol, and water) with
3 × 150 mL of each solvent under sonication for 30 min. Each extract was concentrated under
vacuum and dried under nitrogen stream until complete evaporation of solvent. The extractions
yielded 0.52 g of hexane (5.2%), 0.52 g of ethyl acetate (5.2%), 1.12 g of methanol (11.1%),
and 0.60 g of water (5.9%) extracts. The same extraction procedure was performed on Z.
officinale rhizome powder (10 g) to yield 0.27 g of hexane (2.7 %), 0.12 g of ethyl acetate (1.2
%), 0.50 g of methanol (5 %), and 0.36 g of water (3.6 %) extracts. In addition, the two species
were extracted separately with methanol (called “full methanol extract”) for metabolite
comparison.
HPLC-UV-ELSD analysis. Extracts were analyzed by HPLC with UV and ELSD detection
on a X-Bridge C18 column (250 × 4.6 mm, i.d., 5 µm; Waters, Milford, MA, USA) using a
mobile phase consisting of water (A) and methanol (B) containing both 0.1% formic acid;
analysis was performed with linear gradient from 5 to 95% of B in 60 min. The samples were
diluted in methanol at 5 mg/mL (injection of 10 µL).
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Fractionation and isolation of constituents. The optimized analytical HPLC-UV-ELSD
conditions were geometrically transferred to semi-preparative conditions to ensure the same
selectivity for fractionation of the hexane extract of Z. purpureum (75 mg repeated 4 times).
The hexane extract was dissolved in methanol and filtered to remove precipitate prior
fractionation. Fractionation was performed on a C18 column (250 × 19 mm, i.d., 5 m; Waters,
Milford, MA, USA) with water (A) and methanol (B), containing both 0.1% formic acid as
mobile phase, and using a linear gradient from 5 to 100% B in 40 min. The flow rate was fixed
at 10 mL/min and the UV detection was set at 254, 260, 280 and 366 nm. All collected fractions
were concentrated under vacuum and dried under nitrogen. Using this method, fraction A
yielded 1 and 2 (4.1 mg), B yielded 3 and 4 (1.5 mg), C yielded 5 (1.0 mg), D yielded 6 (57.3
mg), E yielded 7 (46.3 mg), H yielded 8 (54.4 mg), I yielded 9 (2.9 mg), L yielded 10, 11 and
12 (7.7 mg), M yielded 11 and 12 (68 mg).
Synthesis of banglene. The synthesis of banglene has been the subject of previous studies in
our group8, 23 and the conditions have been improved in the present work. A mixture of (E)-4-
(buta-1,3-diene-1-yl)-1,2-dimethoxybenzene (2.0 g, 11.2 mmol) and p-hydroquinone (35.8 mg,
325 μmol) in p-xylene (5.0 mL) was stirred for 12 h at 140 °C under argon. The solution was
cooled down to room temperature and concentrated in vacuo. The mixture was purified by
silica-gel column chromatography (hexane : ethyl acetate = 3:1) to give banglene (1.7 g, 79%)
as a 1:1 diastereomeric mixture (11, 12). The aliquot (1.0 g) of the mixture (11, 12) was
subjected to silica-gel column chromatography (Biotage SNAP Ultra 25g, hexane (A) – ethyl
acetate (B)) and eluted with a gradient of 5 to 40% B for 10 column volumes, to give 11 (100
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mg) and 12 (100 mg). These yields were sufficient to evaluate the compounds in the zebrafish
epilepsy assay only.
[푎]2퐷7
Trans-banglene (synthesized compound):
 0.0 (c. 0.22, CHCl3). 1H NMR (CDCl3, 500
MHz) δ 1.67 (1H, qd, J = 12.8, 10.0, 4.4 Hz, H-5b), 1.92 (1H, dq, J = 12.8, 4.4 Hz, H-5a), 2.22
(2H, m, H-6), 2.35 (1H, qd, J = 10.0, 8.7, 7.3, 2.9 Hz, H-4), 3.18 (1H, dt, J = 8.7, 2.9 Hz, H-
3), 3.82 (3H, s, 3'OMe), 3.85 (3H, s, 4'OMe or 6''OMe), 3.86 (3H, s, 4'OMe or 6''OMe), 3.87
(3H, s, 5''OMe), 5.68 (1H, dt, J = 9.9, 2.9, 2.0 Hz, H-2), 5.90 (1H, dq, J = 9.9, 3.3, 2.9 Hz, H-
1), 6.02 (1H, dd, J = 16.0, 7.3 Hz, H-1''), 6.09 (1H, d, J = 16.0 Hz, H-2''), 6.70 (1H, d, J = 1.7
Hz, H-2'), 6.73 (1H, dd, J = 8.2, 1.7 Hz, H-6'), 6.78 (3H, m, H-5', H-7'', H-8''), 6.81 (1H, d, J =
2.3 Hz, H-4''); 13C NMR (CDCl3, 126 MHz) δ 24.5 (C-6), 27.8 (C-5), 45.4 (C-4), 48.0 (C-3),
55.8, 55.9, 55.9 (3'OMe, 4'OMe, 5''OMe, 6''OMe), 108.7 (C-4''), 110.8 (C-5'), 111.2 (C-7''),
111.7 (C-2'), 118.8 (C-8''), 120.4 (C-6'), 127.6 (C-1), 128.9 (C-2''), 130.3 (C-2), 131.0 (C-3''),
132.2 (C-1''), 137.6 (C-1'), 147.3 (C-4'), 148.3 (C-6''), 148.6 (C-3'), 148.9 (C-5''); ESI(+)-
+
HRMS m/z 398.2324 [M+NH4]+ (calcd. for C24H29O4NH3 , 398.2326, Δppm = 1.5).
[푎]2퐷7
Cis-banglene (synthesized compound):
 0.0 (c. 0.70, CHCl3). 1H NMR (CDCl3, 500
MHz) δ 1.6 (2H, m, H-5), 2.2 (2H, m, H-6), 2.7 (1H, tt, J = 9.6, 9.2, 3.5 Hz, H-4), 3.5 (1H, t, J
= 3.5, 2.1 Hz, H-3), 3.8 (3H, s, 3'OMe), 3.8 (3H, s, 5''OMe), 3.9 (3H, s, 6''OMe), 3.9 (3H, s,
4'OMe), 5.6 (1H, dd, J = 15.8, 9.2 Hz, H-1''), 5.8 (1H, dt, J = 10.0, 4.6, 2.2 Hz, H-2), 6.0 (1H,
dq, J = 10.0, 2.1 Hz, H-1), 6.3 (1H, d, J = 15.8 Hz, H-2''), 6.7 (1H, d, J = 1.9 Hz, H-2'), 6.7
(4H, m, H-6', H-4'', H-7'', H-8''), 6.8 (1H, d, J = 8.2 Hz, H-5'); 13C NMR (CDCl3, 126 MHz) δ
24.3 (C-5), 24.8 (C-6), 42.6 (C-4), 45.8 (C-3), 55.8, 55.9 (3'OMe, 4'OMe, 5''OMe, 6''OMe),
108.7 (C-4''), 110.3 (C-5'), 111.2 (C-7''), 113.6 (C-2'), 118.7 (C-8''), 122.0 (C-6'), 128.1 (C-1),
128.5 (C-2''), 129.2 (C-2), 131.1 (C-3''), 132.5 (C-1''), 133.8 (C-1'), 147.5 (C-4'), 148.1 (C-3'),
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148.2 (C-6''), 148.9 (C-5''); ESI(+)-HRMS m/z 398.2325 [M+NH4]+ (calcd. for C24H29O4NH3 ,
398.2326, Δppm = 1.3).
Experimental animals. Adult zebrafish (Danio rerio, Tübingen strain) were maintained in a
14/10 h light/dark cycle with standard aquaculture conditions: 26 °C, pH 7.5, conductivity 500
μS. Embryos obtained with natural mating were raised in E3 embryo medium (5 mM NaCl,
0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) supplemented with 0.0001% methylene blue,
at 28.5 °C until four days post-fertilization (dpf). Larvae were assessed visually and manually
sorted to maintain only larvae with normal 4 dpf morphology, including swim bladder inflation.
Experimentation with zebrafish larvae was performed before 6 dpf. Up to this developmental
age, in agreement with Swiss guidelines and the European Parliament directive 2010/63/EU,
larvae do not independently feed, and are not subject to experimental restrictions.
Male 4-week-old ICR mice (body weight 20-24 g) were purchased from Japan SLC (Shizuoka,
Japan). Animals were maintained in an air-conditioned room with controlled temperature (22
± 2 °C) under a 12/12 h light/dark cycle. Mice were housed in standard-sized plastic cages with
sawdust (4-6 mice per cage), allowed water ad libitum, fed standard commercial pellets, except
during experiments. The experimental protocol was in compliance with the “Guide for the Care
and Use of Laboratory Animals”24 and was conducted under the guidelines of the animal
research committee of the University of Tokushima graduate school under the approval number
T30-85.
Determination of MTC in zebrafish larvae. A maximum tolerated concentration (MTC) was
set to determine the range of appropriate sample concentrations for the assays, without signs
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of toxicity. Zebrafish larvae of 4 dpf were incubated in different sample concentrations for 18
h in 1 mL of embryo medium (final DMSO concentration of 1% w/v). Five to 10 replicates
were used for each concentration and the assay was repeated at least once. The behavioral
profile of larvae was examined carefully to identify, in comparison to the control group,
toxicity signs such as loss of posture, hypoactivity, no touch response, morphology changes or
death.14 Thus, the MTC of a sample was determined as the highest concentration causing no
signs of toxicity in all replicates.
Evaluation of antiseizure activity. Zebrafish larvae of 5 dpf were monitored in a dark
chamber under light conditions (Fig. S1, Supporting Information). The setup consisted of a
halogen lamp (20 W), a tripod, a white background to place the 96 well-plate on, and a
Panasonic 4K HC-WX979 full HD camcorder to record larval movements from a top view
within a defined time period (30 min in the experimental set-up of the present study).
A computer equipped with an Intel® CoreTM i5-6500T CPU, an Intel® HD Graphics 530
(Skylake GT2), 8 Go RAM, and 500 Go hard drive was used for data acquisition. The larval
locomotor activity was quantified with the python-based PyTracker open-source automated
tracking software20 installed on a Linux system (Ubuntu 18.04.3 LTS, Gnome 3.28.2, 64 bits)
(Fig. S2, Supporting Information). Two modifications were made to the source code. The first
was to calculate the average movements of each region of interest (ROI) during the time of
analysis, and the second was to export the raw data in .csv format. The new source code is
available as supplementary material in the repository folder under the name
PyTracker_Zebrafish_TBrillatz.zip.
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Sixty zebrafish larvae were placed in a flat bottom 96-well plate (Corning®, Corning, NY,
USA) (1 larvae/well, at least five replicates for each concentration tested, e.g. 3, 10, 30, 50,
100 µg/mL for extracts/fractions and 8, 25, 50 µM for the isolated compounds) and incubated
at 28 °C for 18 h as performed in Afrikanova et al.14 Larvae medium added to DMSO (1% w/v)
was used as the vehicle control. PTZ added to the vehicle at 20 mM served as positive control.
The toxicity of some solvents in 6-7 dpf larvae was determined previously.25 PTZ added to the
medium elicits distinct seizure-like behaviors characterized by greatly increased locomotor
activity (Stage I) and “whirlpool-like” circling swim behavior (Stage II). Thus, normal
locomotor activity and seizures could be differentiated by seeing a significant increased ratio
between movement with vehicle only and vehicle with PTZ (Fig. S3, Supporting Information).
Movements of fish larvae were recorded for 30 min after the incubation period. A second
video-tracking of 30 min was recorded exactly 5 min after the addition of PTZ solution. The
sum of the pixel shifts of each ROIs was calculated by the PyTracker for a 30 min period.
Results were normalized as an average percentage compared to the positive control and plotted
as bar and movement/time graphs. Controls were included in each screening to ensure that the
set-up of the experiment does not influence fish larval behavior.
Mouse PTZ seizure model. The antiseizure effect of banglene (50/50 ratio of trans-banglene
(11) and cis-banglene (12)) was evaluated in a mouse PTZ seizure model. The mice were
randomized into three groups (n = 8-11) with two treatment groups which received banglene
at 20 and 50 mg/kg per os (p.o.) respectively, and a control group which received vehicle p.o.
(saline water, equal volume to treatment) every day for five days. The doses of banglene and
its route of administration were determined on the basis of our previous study in which
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banglene was administered orally to mice at 20 and 50 mg/kg with no observed toxicity.8 PTZ
(70ꢀmg/kg) was administered intraperitoneally to animals 60 min after the last administration
of banglene and the seizure level was evaluated during 20 min after the administration of PTZ
with the following score: 0 = no response, 1 = ear and facial twitching, 2 = myoclonic jerks of
the forelimbs without rearing, 3 = myoclonic jerks of the forelimbs and rearing, 4 = turn over
into side position and clonic-tonic seizures, 5 = turn over into back position and generalized
clonic-tonic seizures.26
UHPLC-HRMS/MS analysis. The extracts were analyzed by Ultra-high performance liquid
chromatography (UHPLC)-HRMS/MS for the metabolite profiling of Z. purpureum and Z.
officinale. The HRESI-II parameters were as follows: source voltage: 3.5 kV (pos); sheath gas
flow rate (N2): 50 units; auxiliary gas flow rate: 12.5 units; spare gas flow rate: 2.5; capillary
temperature: 262.5 ˚C; S-Lens RF Level: 45. The data-dependent MS/MS events were
performed on the three most intense ions detected in full scan MS (Top3 experiment). The
MS/MS isolation window width was 1 Da, and the stepped normalized collision energy was
set to 15, 30, and 45 units. In data-dependent MS/MS experiments, full scans were acquired at
a resolution of 35,000 FWHM (at m/z 200) and MS/MS scans at 17,500 FWHM both with an
automatically determined maximum injection time. After acquisition in a MS/MS scan, parent
ions were placed in a dynamic exclusion list for 2.0 s. The UHPLC conditions were as follows:
BEH C18 column (100 × 2.1 mm, 1.7 m; Waters, Milford, MA, USA) using a mobile phase
consisting of water (A) and methanol (B) containing both 0.1% formic acid; flow rate, 400
L/min; linear gradient of 5 to 100% of B in 30 min and held during for 4 min; Injection
volume, 2 L; samples diluted in methanol at 5 mg/mL.
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Molecular network analysis. The MS data were converted from .raw (Thermo) standard data
format to .mzXML format using the MSConvert software, part of the ProteoWizard package.27
The molecular network (MN) of MS/MS spectra of Zingiberaceae species was generated using
the online workflow of the Global Natural Products Social Molecular Networking (GNPS).
The converted data (.mzXML) were uploaded onto the GNPS platform. A network was then
created where edges were filtered to have cosine score above 0.7 and more than six matched
peaks. Further edges between two nodes were kept in the network if each of the nodes appeared
in each other’s respective top 10 most similar nodes. The output was visualized using
Cytoscape 3.6 software.28 The GNPS job parameters and resulting data are available at the
following
address
(https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=39384e9ecbbc4cf9970b28f6ca9c1471).
298 Metabolite annotation. The MS data were converted from .raw (Thermo) standard data format
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to .abf format using the Reifycs Abf Converter software (available at
https://www.reifycs.com/AbfConverter/). The converted data (.abf) were treated using the MS-
DIAL software v.2.4.29 The parameters were adjusted as follows: Adducts: [M+H]+, [M+NH4]+,
[M+Na]+; Mass accuracy: MS1 tolerance: 0.01 Da, MS2 tolerance: 0.05 Da; Retention time:
0-30 min; Mass range: 120-1500 Da; Maximum charge number: 2; Minimum peak height:
10000000 amplitude; Mass slice width: 0.1 Da; Sigma window value: 0.5; MS/MS abundance
cut off: 0. After data treatment, all peak features were imported to the MS-FINDER software,30
which is linked with MS-DIAL, and then a comprehensive annotation was performed on MS-
FINDER. The parameters were adjusted as follows: mass tolerance setting: 0.01 Da (MS1),
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0.025 Da (MS2); relative abundance cut off: 5%; formula finder settings: LEWIS and SENIOR
check (yes); isotopic ratio tolerance: 20%; element probability check (yes); element selection
(O, N). Structure Finder settings: tree depth: 2; maximum report number: 100; data sources:
FoodDB, PlantCyc, ChEBI, T3TDB, DrugBank, STOFF, KNApSAcK, NANPDB, PubChem,
UNPD.
Statistical analysis. Data were expressed as mean ± SEM (n ≥ 5). Statistical differences among
the data were assessed by one-way or two-way ANOVA with Dunett’s test for zebrafish assays,
and Steel test and Fischer’s exact tests for mouse assay. Data were compared with positive
control, with P values of < 0.05 (*), < 0.01 (**), < 0.001 (***) and < 0.0001 (****) respectively.
Analyses were performed using Prism v8.3.0 (GraphPad Software).
320 Results and discussion
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Zebrafish antiseizure screening assay. In order to screen the antiseizure activity of the
various extracts of Z. purpureum, a streamlined and cost-effective zebrafish epilepsy assay was
adapted from the zebrafish PTZ-induced seizure model (Fig. S1, Supporting Information).14
A python-based tracking software called PyTracker was used for the calculation of zebrafish
larval locomotor activity (Fig. S2, Supporting Information). This open-source motion-tracking
software is well developed and provides cost-effective solutions compared to commercial
packages for simple and easy measurement of behavioral phenotypes.20-22 The source code of
the software was adapted to calculate simultaneously the average movements of each regions
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of interest (ROIs) and to export data in .csv format.20 To date, no open-source motion-tracking
software have been reported for zebrafish-based analysis of antiseizure natural products.
In this study, a standard HD camcorder was used to record larval movements, although an
infrared camera is generally recommended to avoid light-induced behavioral phenotypes in
zebrafish.31 Zebrafish larvae have been reported to become light-sensitive at 5 days post-
fertilization (dpf),32 with time of day and light conditions also influencing their locomotion.33
However, the antiseizure assay performed in this study under illumination with zebrafish of
4-5 dpf showed that light-induced behavior was negligible compared to larvae treated with
pentylenetetrazole (PTZ) (Fig. S3, Supporting Information). In these experiments, the
behavioral profile was recorded for zebrafish larvae exposed to: vehicle, vehicle with PTZ, and
finally the ASD topiramate (TPR) added to PTZ. The results demonstrated that the locomotor
activity of zebrafish exposed to PTZ exhibited the same behavioral profile as that described by
Afrikanova et al (Fig. S3, Supporting Information).14 In addition, zebrafish treated with TPR
showed similar seizure reduction activity (Fig. S4, Supporting Information). Since the assays
performed with the vehicle, PTZ and TPR were comparable to those of Afrikanova et al.,14 this
streamlined zebrafish seizure assay was used for the present study. PTZ and TPR were included
in each bioassay to ensure that the experimental set-up does not influence zebrafish larval
behavior.
Zebrafish antiseizure screening of Z. purpureum. The rhizome powder of Z. purpureum was
extracted by solvents of increasing polarity (hexane, ethyl acetate, methanol, and water) and
the extracts were submitted to the zebrafish antiseizure assay (Fig. 1). The hexane extract
showed the strongest antiseizure activity, with a reduction of PTZ-induced locomotor activity
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at 30 μg/mL (Fig. 1A-B). On the other hand, ethyl acetate, methanol, and aqueous extracts
were not active at the tested concentrations. The hexane extract was evaluated in an additional
independent experiment to confirm the antiseizure activity, where it reduced 75% of PTZ-
induced locomotor activity (50 μg/mL, p < 0.0001) (Fig. S5, Supporting Information).
Therefore, a bioactivity-guided isolation protocol was conducted to isolate the compounds
responsible for the antiseizure activity of the hexane extract of Z. purpureum.
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369
Fig. 1. Effect of the Z. purpureum extracts on larval locomotor activity. (A) Hexane, ethyl
acetate, methanol, aqueous extracts were tested at 10, 30 and 100 μg/mL. (B) PTZ-induced
locomotor activity curves for a 30 min tracking period with 5 min time periods after 18 h
pretreatement with the hexane extract. PTZ was used at 20 mM as proconvulsant agent (black
bar/curve). Controls are described as “-” for the vehicle control and “+” for the positive control.
Data are represented by the average % ± SEM (n = 5) against Veh + PTZ. Statistical analysis
was performed by one-way ANOVA (bar graph) and two-way ANOVA (movement/time
graph) with Dunnett’s test to compare samples with positive control, with P value of < 0.05
(*).
370
Bioassay-guided fractionation and identification of active compounds.
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The hexane extract of Z. purpureum (Fig. S6, Supporting Information) was analyzed by reverse
phase HPLC-UV-ELSD analysis using a gradient of methanol and water. The conditions were
optimized to improve selectivity (Fig. 2A) and a gradient transfer was applied to ensure the
same selectivity for semi-preparative HPLC-UV fractionation (Fig. 2B).34 The fractionation of
the hexane extract yielded 15 fractions (A-O) (Fig. 3B). Fractions A-E, H, I, L, and M have
been unambiguously identified by HRMS and NMR (Fig. S7-61, Supporting Information) and
by comparison with literature as F-A, vanillin (1)35 and cassumunol H (2)36 in mixture; F-B,
ferulaldehyde (3)37 and methyl vanillin (4)38 in mixture; F-C, 3,4-dimethoxycinnamaldehyde
(5)39;
F-D,
(E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol
(6)40;
F-E,
(E)-4-(3,4-
dimethoxyphenyl)but-3-enyl acetate (7)41; F-H, (E)-1-(3,4-dimethoxyphenyl)buta-1,3-diene
(8)42; F-I, (E)-1-(3,4-dimethoxyphenyl)but-1-ene (9)43; F-L, 3,4-bis (3,4-dimethoxyphenyl)-
1,5-octadiene (10)44, 10, and 11 in mixture; F-M, trans-banglene (11)44 and cis-banglene (12)44
in mixture (Fig. 2C). Compounds 1-9, 11, 12 were already described in Z. purpureum, except
for 10 which was described for the first time, although it has been reported as a product after
pyrolysis of cis-1,2-bis[(E)-3,4-dimethoxystyryl]cyclobutane via Cope rearrangement.44
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OCH3
OCH3
OCH3
HO
OH
OH
OCH3
H3CO
H3CO
H
H
O
OCH3
OCH3
H
H
O
O
1
6
2
3
4
8
5
9
O
O
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
O
HO
O
7
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
10
11
12
386
387
Fig. 2. Analytical HPLC-UV chromatogram of the bioactive extract of Z. purpureum, its semi-
preparative fractionation and the structures of the isolated compounds (1-12). (A) Optimized
HPLC-UV chromatogram (254 nm) of the hexane extract of Z. purpureum rhizomes. (B) After
geometrical gradient transfer, the hexane extract was fractionated by semi-preparative HPLC-
UV (254 nm). The fractions collected were labelled (A-O) and the isolated compounds (1-12)
were identified. (C) Structures of the isolated compounds (1-12).
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All fractions were evaluated in the zebrafish seizure assay at 10, 30 and 100 μg/mL and fraction
M gave the strongest antiseizure activity (Fig. S75, Supporting Information). Fraction M was
analyzed by NMR as an equivalent mixture (50/50 ratio) of trans-banglene (11) and cis-
banglene (12). The mixture of trans- (11) and cis-banglene (12), named “banglene”, occurs
naturally in Z. purpureum and has previously been reported to contribute to its neurotrophic
activities.8 Therefore, this fraction was evaluated in an independent experiment at
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concentrations below its maximum tolerated concentration (MTC) and reduced 68% of PTZ-
induced seizures (20 μg/mL, p < 0.0001) (Fig. S76, Supporting Information).
The constituents (11, 12) present in fraction M were synthesized at a larger scale by improving
an existing protocol.8, 23, 45 This yielded a mixture of 11 and 12 and targeted isolation performed
on normal phase yielded pure 11 and 12. The NMR spectrum recorded on each synthesized
compound confirmed that they matched those present in fraction M (Fig. S62-74, S77,
Supporting Information). These compounds (11, 12) were also analyzed separately by chiral
chromatography and showed the presence of two peaks of equal intensity corresponding to 11
and two peaks of equal intensity corresponding to 12 (Fig. S78, Supporting Information). This
data indicated that 11 and 12 occurred as a racemic mixture.
Trans-banglene (11) and cis-banglene (12) were tested separately and 12 demonstrated a
stronger antiseizure activity than 11 with a reduction of 68% of PTZ-induced locomotor
activity (50 μM, p < 0.0001) (Fig. 3). This result suggested that stereochemistry of the
cyclohexene ring substitution is potentially important for the biological activity. In addition,
compounds 11 and 12 demonstrated an antiseizure activity at a concentration four to eight
times lower than topiramate.
To confirm the activity observed in the zebrafish streamlined assay, 11 and 12 were evaluated
in a PTZ mouse seizure model.
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Fig. 3. Effect of trans-banglene (11) and cis-banglene (12) on larval locomotor activity. (A)
Compounds 11 and 12 were tested at concentrations according to their MTC. (B-C) PTZ-
induced locomotor activity curves for a 30 min tracking period with 5 min time periods after
18 h pretreatement with 11 (B) and 12 (C). PTZ was used at 20 mM as proconvulsant agent
(black bar/curve). Controls are described as “-” for the vehicle control and “+” for the positive
control. Data are represented by the average % ± SEM (n = 10-30) against Veh + PTZ.
Statistical analysis was performed by one-way ANOVA (bar graph) and two-way ANOVA
(movement/time graphs) with Dunnett’s test to compare samples with positive control, with P
value of < 0.05 (*), < 0.01 (**), <0.001 (***), <0.0001 (****).
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PTZ mouse seizure model. For the evaluation of the antiseizure activity in rodents, more than
600 mg of the active compounds was necessary. Therefore, trans- (11) and cis-banglene (12)
were synthesized on a large scale from the monomer 8 by Diels-Alder reaction in the presence
of hydroquinone in toluene.8, 23, 45 While 11 and 12 could be isolated at small scale (see
Synthesis of banglene in Materials and Methods), these compounds could however not be
sufficiently resolved in semi-preparative HPLC chromatography.
Since both compounds were active in the zebrafish epilepsy assay separately and in mixture
(fraction M), the 50/50 mixture of isomers 11 and 12, was tested in a PTZ mouse seizure model.
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In this assay, banglene was administered per os at 20 and 50 mg/kg and seizures were induced
by PTZ (70 mg/kg, intraperitoneally). It should be noted that in our previous work, the oral
administration of these doses showed neurotrophic activity with no observed toxic effect and
was sufficient to measure banglene in plasma and brain.8 The seizure level was evaluated
according to a modified Racine’s scale.26 The administration of banglene demonstrated a dose-
dependent inhibition of PTZ-induced epileptogenic seizures and a moderate inhibitory effect
was observed at 50 mg/kg (p < 0.01) (Fig. 4).
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Fig. 4. Effect of banglene (11, 12) on pentylenetetrazole (PTZ)-induced behavioral seizures in
mice. Banglene (50/50 ratio of trans-banglene and cis-banglene, 20 and 50 mg/kg, per os), was
administered 60 min before PTZ injection (70 mg/kg, intraperitoneally). Behavioral seizure
intensity was evaluated with a modified Racine’s score (1-5) 20 min after PTZ administration.
Data are represented by the mean behavioral score ± SEM (n = 8-11) against control (veh +
PTZ). Statistical analysis was performed by Steel test to compare samples with control, with P
value of < 0.01 (**).
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Additionally, PTZ-induced mortality was not observed after banglene treatment, suggesting
that it antagonized both PTZ-induced behavioral seizures and had no significant toxicity at the
tested doses (Table 1). This result is similar to the suppression of mortality by positive control
such as diazepam.46 These findings are consistent with the antiseizure activity of banglene
(fraction M) previously demonstrated in the zebrafish epilepsy assay. This therefore suggests
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the potential of translating the results obtained in zebrafish to mice in the context of the 3Rs
criteria.47
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Table 1. Effect of banglene (11, 12) on pentylenetetrazole (PTZ)-induced seizure mortality in
mice. Banglene (50/50 ratio of trans-banglene and cis-banglene, 20 and 50 mg/kg, per os), was
administered 60 min before PTZ injection (70 mg/kg, intraperitoneally). Mortality was
measured 20 min after PTZ administration. Statistical analysis was performed by Fisher's exact
test to compare samples with control, with P value of < 0.01 (**).
Banglene
Control
n = 8
20 mg/kg
n = 11
50 mg/kg
n = 8
Seizure mortality (died/total)
6/8 (75%)
3/11 (27%)
0/8 (0%)**
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Metabolite profiling of Z. purpureum and Z. officinale extracts.
Since our strategy has enabled the identification of the active principles of Z. purpureum, and
since Z. officinale is the most widely consumed species, we then conducted a UHPLC-
HRMS/MS metabolite profiling to determine the uniqueness of the compounds present in Z.
purpureum.
Full methanolic rhizome extracts of both species that provides a wide coverage of the
metabolites were prepared, analyzed and compared by UHPLC-HRMS/MS. Additionally, the
bioactive hexane extract (Fig. S79, Supporting Information) and ethyl acetate extract of Z.
purpureum were also analyzed in the same way to obtain an extensive metabolite coverage.
The data were visualized by molecular network (MN) which were generated using the Global
Natural Products Social (GNPS) molecular networking method (Fig. 5).48 In this MN, the node
representing each feature were grouped into cluster by similarity of fragmentation patterns.
The nodes of the MN were color-labeled according to their respective extracts as: blue and
light blue for the full methanolic extracts of Z. purpureum and Z. officinale, and red and green
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for the hexane and ethyl acetate extracts of Z. purpureum, respectively. Compound annotation
were performed on the MS/MS spectra using MS-Finder and SIRIUS chemoinformatic tools.30,
49 Molecular formulae of all detected metabolites were deduced after preprocessing by MZmine
and MS-DIAL.29, 50 The resulting molecular network grouped 64,866 nodes into 1,773 clusters
(Fig. 5A).
A. Molecular network
B. Annotated clusters
Isolated
Annotated
3
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0
.3
0
483.198
4
2
4
.3
6
3
5
5
0
.4
6
9
2
8
7
.2
3
7
281.159
5
2
0
.3
3
3
466.411
4
3
6
.4
6
0
5
1
6
3
6
8
.1
5
5
4
3
1
.3
1
6
3
6
8
.2
3
5
376.212
3
9
4
.3
5
3
4
2
2
.3
8
5
6
4
0
.5
3
7
.5
3
1
6
3
8
.5
2
2
3
2
6
8
.1
7
5
4
6
8
.3
9
8
5
8
.6
5
2
4
1
2
7
3
4
4
.2
2
8
5
5
6
.4
4
2
5
4
0
.4
4
8
3
0
7
.2
6
3
295.175
5
2
6
.4
3
2
3
6
2
.2
6
9
449.326
4
3
8
.3
7
9
4
5
2
.3
9
5
5
0
8
.4
5
9
3
6
7
.1
9
403.285
4
0
8
.3
6
8
5
1
0
.4
3
7
3
3
3
.1
9
1
3
8
4
.2
1
7
5
1
1
.3
0
6
436.233
3
6
59.185
6
0
1
0
.4
6
9
6
4
4
.4
9
5
480.427
6
1
2
.5
0
6
2
1
9
.1
7
4
3
9
7
.2
0
1
3
0
3
.1
5
8
2
6
8
.1
5
4
2
9
8
.1
2
7
3
9
5
.1
8
4
5
419.207
5
7
0
.4
5
8
6
5
8
.5
4
9
6
.4
2
1
4
2
.2
8
6
4
8
2
.4
0
6
5
2
4
.4
5
3
6
8
4
.5
6
2
5
3
2
1
.2
4
2
3
7
6
.3
2
2
4
2
8
7
.1
7
4
5
9
.3
4
9
487.379
4
3
2
.2
8
410.245
5
8
4
.4
7
4
5
5
2
.4
8
4
6
8
.4
7
8
2
2
9
.1
9
5
3
5
9
.2
9
5
377.196
.196
8
6
.3
5
9
.3
3
9
8
.2
4
5
4
0
1
478.243
396.239
6
1
4
.4
8
4
6
1
7
2
.5
2
7
3
8
2
.2
1
7
0
0
.5
5
7
1
7
9
.1
4
3
3
3
5
6
.2
4
3
6
2
8
.5
0
452.3
8
4
8
.6
1
4
8
5
2
5
3
8
8
.2
5
4
5
9
6
.5
1
1
2
4
3
.1
3
8
2
3
7
.1
1
2
454.322
7
0
2
.5
3
7
3
0
3
.2
3
2
5
1
2
.2
7
1
4
8
2
.2
6
251.128
7
7
2
.6
1
5
7
4
4
.5
8
8
6
8
.7
3
9
4
5
8
.4
3
5
0
.2
1
7
3
9
9
.2
3
5
3
0
3
.0
8
3
4
3
9
.2
0
8
3
5
3
0
.2
6
6
5
3
4
2
1
3
.175
3
2
8
.2
1
2
2
7
1
6
.5
5
3
1
3
7
.1
3
2
335.0
476.285
2
7
1
.2
4
2
4
2
3
.3
6
2
3
7
0
.2
7
2
8
.5
8
9
8
9
4
.7
5
5
4
8
8
.3
7
3
3
2
5
.2
3
8
MN1 : Phenylbutenoids
8
3
2
.6
3
7
5
1
3
.5
0
4
5
9
6
.5
4
4
8
6
7
.4
3
1
4
4
1
.3
7
4
1
6
1
.1
3
3
5
2
0
.3
6
4
5
9
.4
0
5
3
5
2
.2
3
3
2
3
5
.0
9
7
97.2
339.159
5
6
4
.3
5
9
4
1
8
5
.3
3
6
8
1
4
9
.1
3
3
5
1
4
.2
8
6
4
0
1
.2
5
1
2
7
3
.1
4
8
2
7
9
3
.1
9
6
3
3
5
.2
5
4
2
7
3
.2
5
8
5
5
4
.4
9
3
2
201.164
4
3
6
.2
1
8
4
0
0
.2
3
8
317.243
0
3
.1
8
2
8
9
.2
1
9
1
.1
0
7
374.196
263.2
3
3
9
.2
1
7
3
0
7
.2
2
7
233.154
4
2
0
.2
2
3
21.201
5
8
0
.5
4
3
5
0
5
4
8
.3
7
4
5
6
7
.5
5
1
9
1
.0
7
434.218
2
0
5
.1
9
5
4
5
1
.3
9
4
5
5
2
.3
6
9
4
9
6
.2
7
5
4
3
0
.2
5
9
192.11
261.185
1
.2
1
6
4
0
1
.2
4
1
289.216
5
8
0
.4
3
4
4
.2
3
4
4
6
6
.2
6
5
2
2
1
.117
2
2
1
.1
9
281.138
520.343
444.312
6
0
2
.5
8
6
5
4
9
.5
4
4
9
4
.2
5
9
6
2
8
.3
1
8
293.17
3
8
0
.2
8
2
9
8
.1
6
5
311.185
377.269
2
9
2
8
6
.201
2
2
0
.9
3
5
336.253
392.28
2
2
0
.9
3
5
359.258
6
1
1
.2
2
2
0
.9
3
5
394.295
4
4
0
.2
4
3
9
349.238
2
2
0
.9
3
5
3
1
9
.2
2
7
2
2
0
.9
3
5
HO M14
M35
405.212
5
8
1
.2
1
5
MN1
366.264
3
8
2
.2
0
7
4
9
.2
1
7
497.327
331.227
3
5
2
.1
6
441.264
OH
Terpenoids
521.254
MN2
538.279
O-Glu
1
2
4
.0
8
7
1
2
3
.0
7
9
3
8
9
.1
1
2
3
7
3
.1
6
5
1
8
1
.0
1
5
1
4
0
.0
6
8
7
2
0
.4
6
8
4
2
5
.2
1
5
4
7
8
.1
7
9
5
3
7
.1
5
1
2
5
8
.2
3
9
4
4
3
.2
7
8
1
2
4
.0
8
7
1
0
7
3
.3
1
4
8
0.157
9
8
1
.2
7
9
9
8
2
.2
7
9
230.248
8
2
3
0
.2
4
1
3
5
.0
7
9
1
2
9
6
.3
7
8
230.248
1
0
5
7
.2
8
5
5
4
.1
7
7
4
6
3
.1
3
5
5
2
.1
9
7
2
3
0
.2
4
8
2
3
4
0
.2
4
8
1
3
4
.0
7
2
1
3
9
.9
8
8
1
3
9
.9
8
8
4
5
9
.4
8
9
2
8
3
1
.2
5
1
3
.1
5
4
2
5
1
2
3
0
.2
4
8
2
3
0
.2
4
8
4
2
5
.269
1
2
4
.0
8
7
5
4
5
0
.2
1
3
1
0
5
8
.2
8
422.217
1
1
4
8
2
.3
.1
3
612.164
1
2
2
.0
7
2
1
6
6
.1
0
9
1
3
9
.9
8
8
5
9
6
.1
6
9
8
3
3
.2
4
1
6
8
6
.1
8
2
230.24
1
2
6
8
.1
0
3
.1
1
8
2
3
0
.2
4
8
2
8
30.248
4
3
8
.2
4
9
1
4
5
.1
3
4
2
3
0
.2
230.248
1
5
3
.1
1
4
1
0
7
2
.3
1
9
2
4
.2
6
9
230.248
1
7
6
.1
0
6
8
3
7
.2
1
9
9
6
9
2
8
8
2
.1
.2
9
5
5
9
1
4
4
.9
8
2
258.279
1
4
8
.0
8
7
1
5
2
.1
1
8
4
6
2
.1
4
7
9
0
8
.2
5
1
2
3
0
.2
4
8
4
5
3
.3
1
6
9
.1
4
5
9
9
8
.2
8
8
1
4
6
.9
8
493.332
1
3
8
.1
0
3
4
4
5
.1
2
371.101
5
2
5
1
7
0
2
.2
1
3
8
4
8
8
5
.2
7
3
5
1
3
6
.0
8
7
1
2
8
.1
1
1
8
0
.1
2
5
4
5
2
.2
2
8
481.331
1
9
4
.1
4
1
2
5
.9
8
6
5
3
6
.1
6
6
9
2
2
.2
7
9
4
5
2
.2
6
5
5
8
24.249
1
9
5
0
1
.0
9
1
8
7
6
1
0
.1
8
4
5
1
.1
4
7
6
0
.2
0
1
7
7
4
.2
.2
5
3
2
8
6
.3
1
230.24
8
1
6
1
.0
8
2
1
5
4
.1
3
4
1
6
3
.0
9
8
1
8
9
.1
1
4
1
4
0
.1
1
8
6
8
4
.2
0
4
0
0
.2
3
4
0
3
.2
3
2
8
0
2
.2
4
9
3
9
3
.1
9
1
4
1
0
.2
1
7
230.248
561.395
1
8
8
.0
9
3
8
.1
0
9
6
7
0
.1
8
8
1
0
5
6
8
4
3
4
.2
1
7
495.347
1
7
4
.0
9
1
5
0
.1
0
3
1
9
2
.1
2
5
7
4
4
.2
0
6
4
3
5
.2
0
1
5
9
6
.4
6
8
5
0
8
9
.3
6
4
5
8
1
8
.2
9
2
5
5
5
7
.1
7
5
7
7
6
.2
5
5
8
.3
4
3
5
5
1
.4
1
3
2
0
2
.1
0
9
4
6
6
.2
4
4
4
2
4
.2
3
3
4
1
7
7
.1
9
5
3
7
8
.3
9
5
5
6
5
.4
2
554.421
1
4
7
5
8
.2
2
1
6
2
6
.2
1
7
8
3
4
.2
2
1
4
9
4.238
1
4
8
.9
7
7
1
7
1
.9
9
3
1
6
5
.1
1
7
9
.1
2
9
9
0
6
.2
6
5
8
6
.3
7
4
4
7
7
.2
1
2
1
5
4
.9
9
202.217
1
7
5
.0
9
8
7
3
0
.1
4
3
7
.2
4
3
1
5
4
.9
9
2
3
0
.2
4
8
6
3
7
.4
1
1
MeO
OMe
1
.1
0
9
1
6
7
.0
1
3
8
9
2
.2
4
4
4
4
8
.2
3
3
2
7
2
.2
5
9
5
7
9
.4
4
1
4
3
1
.348
8
3
2
.2
4
1
8
7
9
4
3
.2
0
6
5
2
.3
0
1
4
549.394
523.38
2
0
0
.0
9
3
4
4
9
.2
3
5
4
1
.3
1
6
1
1
5
4
.9
9
1
5
4
.9
9
6
0
8
.4
6
8
5
3
9
1
.4
1
5
0
7
8
.2
5
4
340.321
1
6
7
.1
2
9
9
8
0
.2
7
8
8
7
8
0
4
.2
0
9
4
8
0
.2
2
3
5
1
0
.2
3
4
1
5
4
.9
9
1
6
7
.0
1
3
1
6
7
.0
1
3
8
.2
2
9
5
4
3
.2
9
4
1
7
2
.9
5
7
1
5
5
4
4
.9
9
5
1
.3
7
9
5
3
5
.3
7
9
3
9
9
.2
5
1
4
0
7
.2
0
1
5
4
.9
9
1
5
4
.9
9
154.99
1
7
9
.0
9
3
4
9
1
.2
2
7
0
8
.2
0
7
3
5
1
.2
8
4
2
3
0
.2
4
8
2
7
2
.2
5
8
4
2
7
.2
8
5
5
.1
9
6
1
5
4
.9
9
4
5
5
.3
6
1
6
7
.0
9
3
1
6
7
.0
1
3
4
6
4
.2
2
8
1
5
4
.9
9
2
8
5
.4
0
5
5
0
.2
6
7
1
6
7
.0
1
3
1
7
6
7
.0
1
3
1
5
4
.9
9
3
1
5
4
.9
9
5
8
4
.4
6
9
5
0
1
.3
5
6
1
6
7
.0
1
3
3
7
1
.1
8
5
4
9
.2
9
1
1
.9
9
5
507.274
2
4
4
.2
6
4
5
6
.4
3
7
5
OMe
OMe
2
1
2
.9
8
3
1
1
6
3
.9
9
1
1
9
6
.0
1
7
2
4
4
.2
2
7
552.321
1
6
.0
1
3
2
7
2
.2
9
4
1
6
7
.0
1
3
1
4
0
5
.1
9
1
4
7
2
.3
5
4
3
MeO
1
4
9
.0
8
2
525.286
6
7
.0
1
3
4
2
2
.2
1
7
0
0
.3
7
4
3
0
5
.0
7
1
9
.9
9
4
1
6
7
.0
1
3
5
0
5
.2
5
8
10
1
6
7
.0
7
387.181
1
3
8
8
.9
9
5
26.39
1
2
5
.9
8
6
3
1
6
.3
2
1
2
8
1
8
.2
9
4
5
8
4
.2
7
1
5
9
.9
6
141.959
9
1
6
7
.0
1
3
4
2
2
.2
5
4
288.922
2
7
4
.2
7
4
3
3
0
.3
3
5
OMe
OMe
1
2
5
.9
8
6
1
7
6
7
.0
1
3
1
2
5
.9
8
6
1
8
0
.9
9
1
2
8
.985
1
6
7
.0
1
3
139.018
167.013
2
2
4
.0
1
2
2
4
6
.2
4
3
2
1
8
.2
2
1
6
7
.0
1
3
Fatty acids
1
7
4
3
.9
9
1
7
6
.0
.0
13
167.013
1
5
4
.9
9
1
6
7
.0
1
3
1
6
7
.0
1
3
Aromatics
1
242.035
183.101
1
3
6
.0
1
3
1
6
7
.0
1
3
2
0
1
.0
0
1
6
7
.1
0
7
1
6
7
.0
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9
4
.2
6
3
2
0
7
.1
3
8
3
5
3
.2
0
9
2
7
5
.2
5
8
3
1
6
.1
7
5
4
6
6
.2
6
5
4
2
8
.2
8
8
1
9
9
.0
6
5
6
1
.2
8
5
4
1
8
.2
2
2
4
5
0
.2
9
6
4
3
7
.1
9
4
5
2
1
.3
2
4
4
3
4
.2
1
8
3
7
8
.2
6
3
3
2
8
.1
9
1
3
4
6
.1
8
6
2
4
1
.1
0
7
2
0
3
.0
5
3
8
.2
6
3
8
6
.2
4
4
1
8
4
.0
7
3
2
6
7
.1
7
2
2
6
8
.1
3
9
8
7
1
.5
7
4
4
2
3
.1
7
8
3
6
4
.3
0
7
6
3
7
.4
9
5
6
0
4
.2
9
1
4
6
3
.2
4
6
3
4
3
.1
5
4
5
7
7
.2
4
3
1
2
8
.0
1
9
4
0
8
.2
0
2
2
4
4
.0
8
7
1
9
6
.1
7
5
2
9
.2
4
5
4
1
3
.2
2
2
4
4
8
.2
3
7
5
4
3
.3
2
M34
OMe
Z. purpureum full MeOH ext.
Z. purpureum Hex ext.
Z. purpureum EtOAc ext.
Z. officinale full MeOH ext.
MeOH blank
OH
OH
645.27
M18
M36
OMe
632.287
O
OH
M26
615.259
OMe
763.311
OMe
MN4 : Shogaols
591.223
747.317
395.149
390.191
781.322
575.227
573.212
557.217
543.203
O
OH
448.343
721.301
369.169
561.213
601.242
396.312
O
340.248
O
OH
587.226
369.134
749.332
OMe
719.284
559.232
368.28
HO
OH
559.234
312.217
766.36
545.217
589.243
571.233
OMe
OMe
OH
HO
284.186
323.221
OH
545.217
M20
604.291
351.253
378.192
OMe
OMe
M22
779.343
O
362.196
505.259
575.227
OMe
695.358
589.242
M30
M23
OMe
277.18
OH
OH
M32
333.242
305.211
O
OH
OMe
589.281
619.252
327.159
OMe
249.149
OH
MeO
O
OH
399.144
M25
361.275
M28
OMe
OMe
488
489
490
491
492
493
494
495
496
Fig. 5. Differential metabolite profiling of Z. purpureum and Z. officinale with isolated
compounds (1-12) and annotation of dereplicated metabolites (M13-M36). (A) Molecular
network. (B) Four annotated clusters: MN1 are phenylbutenoids, MN2 are gingerols, MN3 are
curcuminoids, MN4 are shogaols.Nodes are color-coded in blue for Z. purpureum full methanol
extract, red for Z. purpureum hexane (Hex) extract, green for Z. purpureum ethyl acetate
(EtOAc) extract and light blue for Z. officinale full methanol extract. Purple squares with black
frame are isolated compounds; purple squares with pink frame are annotated compounds.
497
498
All isolated standards (1-12) could be assigned to their specific nodes and none of them were
detected in Z. officinale. The absence of active compounds 11 and 12, notably, confirmed the
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difference in bioactivity measured in both species. The candidate structures were subsequently
restricted to Zingiber genus in order to refine the possibilities of annotation. The MN was used
to propagate the annotation based on the unambiguously identified nodes 1-12 and allowed the
dereplication of 12 metabolites in Z. purpureum (M15-17, M21-22, M26-29, M31, M33, M36).
Four clusters (MN1-MN4) containing characteristic ginger compounds were selected for in-
depth analysis and several annotations were proposed (Fig. 5B). Metabolites 7, 8, 10, M21,
M24, M27, M29, M31, M33 of MN1 and M16 have been annotated as phenylbutenoid
derivatives with C6-C4 units and, to date, these compounds have only been found in
Zingiberaceae families Z. purpureum9, 36, 44, 51-52 and Alpinia flabellata.53 On another hand,
compound M15 has been annotated as a benzoyl derivative which is common in Apiaceae,54
Acoraceae55 and Aristolochiaceae56 genera, and compound M17 was annotated as a cinnamic
acid ester which is present in Zingiberaceae57 and Polygalaceae species.58 In addition, cluster
MN3 was attributed to curcuminoid metabolites (M22, M26, M28) which are mainly found in
Z. purpureum ethyl acetate extract and were most probably not active. The same network has
shown that very few clusters are common between the two species. For example, vanillin
derivatives (1 and 4) and terpenoids (M13, M14, M19, M35) were present in both species (Fig.
5A), whereas aromatics, fatty acids, gingerols (M18, M23, M30, M34, M36) from MN2 and
shogaols (M20, M25, M32) from MN4 were present only in Z. officinale and were therefore
specific to this species (Fig. 5B, Table 2). Overall, the experimental results showed that the
chemical diversity of the two ginger species was significantly different even though they
belong to the same genus.
The MN also revealed that banglene (11, 12) was not present in the full methanol extract of Z.
officinale, which suggested that no antiseizure activity should be observed in this species. To
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confirm this hypothesis, Z. officinale rhizomes were extracted with solvents of increasing
polarity and evaluated in the zebrafish epilepsy assay (Fig. S80, Supporting Information). As
expected by the absence of banglene in the MN, no antiseizure activity was detected in Z.
officinale extracts at 10, 30 or 100 μg/mL.
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Table 2. Compounds annotated by UHPLC-HRMS/MS ESI(+) in the full methanol extracts of Z. purpureum and Z. officinale extracts and in the
hexane and ethyl acetate extracts of Z. purpureum. Isolated compounds 1-12, annotated metabolites M13-M36. Names of annotated metabolites
based on 2D structure. ZP = Zingiber purpureum; ZO = Zingiber officinale; EtOAc = ethyl acetate; MeOH = methanol; ND = Not determined; a =
annotated by SIRIUS.
Presence in
RT
Chemical
Formula
m/z and ionization
Error
(ppm)
MS-Finder
N°
Annotated compounds
ZP
ZP
ZP
ZO
(min)
mode
score (max=10)
Hexane EtOAc MeOH MeOH
M13 4.25
M14 5.07
p-menth-5-ene-1,2-diol
8-hydroxygeranil-8-O-glucoside
Vanillin
C10H18O2
C16H28O7
C8H8O3
171.1379 [M+H]+
333.1908 [M+H]+
153.0547 [M+H]+
225.1122 [M+H]+
179.0703 [M+H]+
167.0705 [M+H]+
197.0810 [M+H]+
209.1178 [M+H]+
193.0863 [M+H]+
239.1277 [M+H]+
207.1019 [M+H] +
0.33
-0.06
-0.5
-0.3
-0.2
-1.4
-1.4
-2.8
-2.0
1.2
✓
✕
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✕
✓
✓
✓
✓
✓
✓
✓
✓
✓
✕
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✕
✕
✓
✕
✕
✕
✕
✕
7.14
4.68
7.44
7.01
8.16
7.74
7.05
7.43
7.44
7.13
6.88
1
2
3
4
5.51
6.08
7.19
7.50
Cassumunol H
C12H16O4
C10H10O3
C9H10O3
C10H12O4
C12H16O3
C11H12O3
C13H18O4
C12H14O3
Ferulaldehyde
Methyl vanillin
M15 8.45
2,4,5-trimethoxybenzaldehyde
(E)-4-(3,4-dimethoxyphenyl)but-3-en-1-ol
3,4-dimethoxycinnamaldehyde
Cassumunol D
6
5
9.10
9.25
M16 9.55
M17 9.88
Ethyl trans
-p-methoxycinnamate
-1.6
M18 10.12
M19 13.1
Hexahydrocurcumin
C21H26O6
C15H24O
C14H18O4
375.181[M+H]+
-2.1
✕
✓
✓
✕
✕
✓
✕
✕
✓
✓
✓
✕
6.53
7.05
6.69
3(15),4,10-bisabolatrien-2-ol
221.1900 [M+H]+
-0.03
-0.5
7
13.57
(E)-4-(3,4-dimethoxyphenyl)but-3-enyl acetate
251.1279 [M+H]+
268.1543 [M+NH4]+
M20 13.84
M21 14.36
8-shogaol
C17H24O3
C24H30O5
277.1797 [M+H]+
0.4
✕
✓
✕
✓
✕
✓
✓
✕
6.69
ND
Banglenol Aa
416.2438 [M+NH4]+
-3.4
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M22 15.09
15.56
Curcumin
C21H20O6
C12H14O2
C19H30O5
C13H16O3
C12H16O2
C19H28O3
C34H36O9
C26H32O6
369.1342 [M+H]+
191.1068 [M+H]+
339.2168 [M+H]+
221.1175 [M+H]+
193.1225 [M+H]+
305.2106 [M+H]+
589.2423 [M+H]+
458.2535 [M+NH4]+
-2.5
-0.8
-0.6
-1.3
-1.0
1.7
✓
✓
✕
✓
✓
✕
✓
✓
✓
✕
✓
✕
✕
✓
✓
✓
✕
✓
✕
✕
✓
✕
✕
✓
✕
✕
✓
✕
7.32
6.68
6.84
ND
8
(E)-1-(3,4-dimethoxyphenyl)buta-1,3-diene
6-gingerol-3-acetate
M23 15.82
M24 15.98
(E)-1-(2,4,5-trimethoxyphenyl)buta-1,3-dienea
(E)-1-(3,4-dimethoxyphenyl)but-1-ene
Cis-8-shogaol
9
16.73
6.47
6.70
6.79
6.08
M25 17.13
M26 17.76
M27 17.93
Cassumunarin C
1.5
Trans-3-(2,4,5-trimethoxyphenyl)-4-[(E)-2,4,5-
trimethoxystyryl]-cyclohexane
3.5
✓
✓
✓
✓
✓
✓
✓
✓
✓
✕
✕
✕
M28 18.10
M29 18.54
Cassumunarin A
C33H34O8
C26H32O6
559.2324 [M+H]+
0.4
3.9
7.07
5.69
Trans-3-(2,4,5-trimethoxyphenyl)-4-[(E)-2,4,5-
trimethoxystyryl]-cyclohexane
458.2531 [M+NH4]+
M30 18.91
M31 19.02
6-gingerdiol-3,5-diacetate
Phlain III
C21H32O6
C24H30O5
C24H28O4
C21H32O3
C25H30O5
398.2453 [M+NH4]+
416.2439 [M+NH4]+
398.2328 [M+NH4]+
333.2425 [M+H]+
22.1
-2.1
-0.6
-0.2
-1.1
✕
✓
✓
✕
✕
✓
✓
✕
✕
✓
✕
✕
✓
✕
✕
✓
6.00
6.94
7.55
6.74
7.26
10
20.12
3,4-bis (3,4-dimethoxyphenyl)-1,5-octadiene
10-shogaol
M32 20.15
M33 20.26
1-[(E)-2-[2-(3,4-dimethoxyphenyl)cyclohex-3-
en-1-yl]ethenyl]-2,4,5-trimethoxybenzene
428.2434 [M+NH4]+
✓
✓
✓
✕
M34 20.43
11/12 20.68
11/12 21.08
M35 21.5
M36 26.58
3’-methoxy-6-gingerdiol-3,5-diacetate
Banglene cis/trans
C22H34O6
C24H28O4
C24H28O4
C15H24
412.2681 [M+NH4]+
398.2324 [M+NH4]+
398.2325 [M+NH4]+
205.1953 [M+H]+
431.3155 [M+H]+
3.2
1.5
1.3
-1.1
0.2
✕
✓
✓
✓
✕
✕
✓
✓
✕
✕
✕
✓
✓
✕
✕
✓
✕
✕
✓
✓
6.17
6.92
6.88
8.91
ND
Banglene cis/trans
Bisabolatriene
3-(3,4-dihydroxyphenyl)propyl (9E,12E)-
C27H42O4
octadeca-9,12-dienoatea
531
532
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In conclusion, this study reports the identification of the antiseizure compounds of Z.
purpureum, a plant commonly used as a spice in Indonesia. The results from both zebrafish
and mouse antiseizure models confirmed the previous assumption on the potential benefits of
Z. purpureum in CNS disorders. To our knowledge, this is the first report of a bioactivity-
guided isolation with an adapted zebrafish epilepsy assay based on low-cost equipment and
open-source automated tracking software which proved to be predictive for rodent assays. Thus,
this platform was well-suited to the identification of the main antiseizure constituents. Besides,
the metabolite profiling analysis of Z. purpureum and Z. officinale showed, with the exception
of vanillin derivatives and terpenoids, that the composition of the two species was different
and that the active compounds were present only in Z. purpureum, which was confirmed
experimentally in the zebrafish epilepsy assay. The detailed processing of these data provided
a comprehensive overview of the chemical composition of Z. purpureum, and this profiling
could be useful for other groups studying Zingiber plants. The identified active compounds
will be tested in other in vivo seizure models and their mechanism of action investigated.
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Author Contributions
549
550
551
Conceptualization, MK, TB, KT, KI, YF and JLW; investigation, MK, TB, KH, ST, NJ, EFQ,
PMA, LM and KT; data curation, MK, TB, and KT; writing-review and editing, MK, TB, KT,
KI, NJ, EFQ, ADC, YF and JLW.
552
553
Acknowledgments
554
555
556
557
558
559
560
561
The authors acknowledge Mr. W. Tohir, chairman of NOFA (National Outstanding Farmer
Association) in Indonesia for his collaboration. The authors thank HOSODA Co. Ltd for
providing the plant material (Z. purpureum). The author (TB) thank Prof. Ian Woods for the
development and free access to the PyTracker. The author (TB) is grateful to Yann Manet and
Dr. Giuseppe Marco Randazzo for the modification of the PyTracker source code. The author
(MK) acknowledges gratefully the Yamada Science Foundation and The Nagai Foundation
Tokyo for their support. The ISPWS (Prof. J-L. Wolfender) is thankful to the SNSF for the
support in the acquisition of the NMR 600 MHz (SNF R’Equip grant 316030_164095).
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563
564
565
Conflicts of Interest
The authors declare no conflict of interest.
566
567
568
569
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571
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Supplementary Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi*****.
Experimental set up of low-cost open-source zebrafish bioassay; behavioral profile of zebrafish
larvae exposed to PTZ; effect of topiramate on larval locomotor activity; results of hexane
extract of Z. purpureum retested in the zebrafish epilepsy assay; HPLC-UV-ELSD
chromatograms of Z. purpureum extracts; NMR and UHPLC-HRMS data of isolated
compounds; larval locomotor activity of the fractions of hexane extract of Z. purpureum; NMR
comparison of fraction M, 11, and 12; chiral HPLC analysis of 11 and 12; UHPLC
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