Biotransformation of pungent constituents from ginger (Zingiber officinale Roscoe) by Colletotrichum gloeosporioides yields oxidative ortho–ortho coupling products

Article abstract of DOI:10.1080/10242422.2021.1878159

This work investigated the biotransformation of ginger constituents (zingerone, [6]-shogaol, [6]-gingerol, and methyl-[6]-gingerol) by the pathogenic fungus Colletotrichum gloeosporioides. Experiments were carried out with and without deuterium-labelled compounds. The product metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry and liquid chromatography solid phase extraction-nuclear magnetic resonance. Substrates supplied to the fungus were incorporated into metabolic pathways mostly by oxidation reactions, including aromatic carbon–carbon coupling. Zingerone and [6]-gingerol biotransformation products included biphenol dimers. A biodegradation pathway for biphenol formation was proposed based on the presence of the intermediate 4-(2-hydroxyethyl)-2-methoxyphenol, commonly identified from [6]-gingerol and [6]-shogaol biodegradation. This intermediate likely originates from a Baeyer–Villiger reaction followed by hydrolysis. The C–C coupling of molecules could result in phenolic oxidative ortho–ortho coupling, suggesting that biphenol dimers are products of C. gloeosporioides laccase catalysis.

Full text of DOI:10.1080/10242422.2021.1878159

Biocatalysis and Biotransformation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Biotransformation of pungent constituents from
ginger (Zingiber officinale Roscoe) by Colletotrichum
gloeosporioides yields oxidative ortho–ortho
coupling products
Roberta Marques Dias de Ávila, Leonardo Toffano, João Batista Fernandes,
Maria Fátima das Graças Fernandes da Silva, Lorena Ramos Freitas de Sousa
& Paulo Cezar Vieira
To cite this article: Roberta Marques Dias de Ávila, Leonardo Toffano, João Batista Fernandes,
Maria Fátima das Graças Fernandes da Silva, Lorena Ramos Freitas de Sousa & Paulo Cezar
Vieira (2021): Biotransformation of pungent constituents from ginger (Zingiberꢀofficinale Roscoe)
by Colletotrichumꢀgloeosporioides yields oxidative ortho–ortho coupling products, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2021.1878159
To link to this article: https://doi.org/10.1080/10242422.2021.1878159
Published online: 14 Feb 2021.
Article views: 58
View supplementary material
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ibab20

BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2021.1878159
REAEARCH ARTICLE
Biotransformation of pungent constituents from ginger (Zingiber officinale
Roscoe) by Colletotrichum gloeosporioides yields oxidative ortho–ortho
coupling products
a
b
a
ꢀ
~
ꢀ
, Maria Fatima das
Roberta Marques Dias de Avila , Leonardo Toffano
, Joao Batista Fernandes
Grac¸as Fernandes da Silvaa , Lorena Ramos Freitas de Sousac
and Paulo Cezar Vieiraa,d
a
b
~
~
Department of Chemistry, Federal University of Sao Carlos, Sao Paulo, Brazil; Department of Agronomy, Brasil University,
c
d
ꢀ
~
Descalvado, Brazil; Special Academic Unit of Chemistry, Federal University of Goias, Catalao, Brazil; School of Pharmaceutical
Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
~
~
~
ABSTRACT
ARTICLE HISTORY
Received 15 June 2020
Revised 6 January 2021
Accepted 13 January 2021
This work investigated the biotransformation of ginger constituents (zingerone, [6]-shogaol, [6]-
gingerol, and methyl-[6]-gingerol) by the pathogenic fungus Colletotrichum gloeosporioides.
Experiments were carried out with and without deuterium-labelled compounds. The product
metabolites were analyzed by liquid chromatography coupled to tandem mass spectrometry
and liquid chromatography solid phase extraction-nuclear magnetic resonance. Substrates sup-
plied to the fungus were incorporated into metabolic pathways mostly by oxidation reactions,
including aromatic carbon–carbon coupling. Zingerone and [6]-gingerol biotransformation prod-
ucts included biphenol dimers. A biodegradation pathway for biphenol formation was proposed
based on the presence of the intermediate 4-(2-hydroxyethyl)-2-methoxyphenol, commonly
identified from [6]-gingerol and [6]-shogaol biodegradation. This intermediate likely originates
from a Baeyer–Villiger reaction followed by hydrolysis. The C–C coupling of molecules could
result in phenolic oxidative ortho–ortho coupling, suggesting that biphenol dimers are products
of C. gloeosporioides laccase catalysis.
KEYWORDS
Biotransformation;
Colletotrichum gloeospor-
ioides; biphenol; ginger
GRAPHICAL ABSTRACT
Introduction
anti-inflammatory activities (Citronberg et al. 2013; Ho
et al. 2013; Mashhadi et al. 2013; Kumar et al. 2014;
Nile and Park 2015).
Zingiber officinale Roscoe (Zingiberaceae family), com-
monly known as ginger, is a root used worldwide as a
food and beverage additive and is also found in trad-
itional medicines for the treatment of several diseases
(Kubra and Rao 2012; Baliga et al. 2013; Villalvilla et al.
2014; Bartels et al. 2015; Prasad and Tyagi 2015;
Semwal et al. 2015; Azimi et al. 2016; Choi et al. 2018;
Kou et al. 2018). Pharmacological properties of ginger
include antioxidative, anticancer, antimicrobial, and
Primary and secondary metabolites have been pre-
viously identified from ginger. The pungent constitu-
ents have been shown to be the major compounds
responsible for the biological activities associated with
ginger (Jolad et al. 2004; Mashhadi et al. 2013; Choi
et al. 2018). Dehydration of [6], [8], and [10]-gingerols
yields the corresponding shogaols from dried ginger
(Bhattarai et al. 2007) that, after hydrogenation by
~ ~ ~
School of Pharmaceutical Sciences of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto
CONTACT Paulo Cezar Vieira
pcvieira@fcfrp.usp.br
14040-903, Brazil
Supplemental data for this article can be accessed here.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
R. M. D. DE ÁVILA ET AL.
microorganisms, become paradols (Stoner 2013). [6]-
Shogaol, one of the main pungent constituents of gin-
ger, has confirmed biological properties, including
antibacterial, anti-hepatotoxic, antioxidative, anti-
cancer, and anti-inflammatory activities (Semwal et al.
2015; Jo et al. 2016; Kou et al. 2018). Heating or drying
ginger root activates the retro-aldol reaction of gin-
gerol, generating zingerone, another compound with
pharmacological potential (Ahmad et al. 2015).
Previously, we investigated the chemical and bio-
logical properties of ginger metabolites, and we found
that [10]-gingerol is a promising compound for the
treatment of triple negative breast cancer (Martin
et al. 2017; Fuzer et al. 2019).
bis(trimethylsilyl)amide solution (CAS 4039-32-1) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
~
Ginger rhizome (6 kg) was purchased locally (Sao Carlos,
SP, Brazil).
[6]-Gingerol was obtained by purification from the
fresh ginger rhizome (Da Silva et al. 2012). Methyl-[6]-
gingerol and [6]-shogaol were derived from [6]-gin-
gerol reactions (Villalvilla et al. 2014). The deuterium
labelling reaction (Tamogami et al. 2016) was per-
formed on [6]-gingerol and zingerone, yielding [6]-gin-
gerol-D3 (54%) and zingerone-D5 (66.3%), respectively.
Further information about purification procedures, syn-
theses, and additional materials (1H NMR and MS data)
are described in the Supplementary Information.
A Shimadzu SCL-10AVP high-performance liquid chro-
matography (HPLC) instrument with an LC-6AD photo-
diode array detector was used for purification (Shimadzu,
Columbia, MD, USA). Structural characterization of com-
pounds was completed using nuclear magnetic resonance
spectroscopy (NMR), gas chromatography-mass spectrom-
etry (GC-MS), HPLC coupled to a triple quadrupole MS
system with electrospray ionization (ESI) (LC-ESI-MS/MS),
and liquid chromatography-solid phase extraction-nuclear
magnetic resonance (LC-SPE-NMR). The instruments used
were: DRX-400 NMR (Bruker, Billerica, MA, USA) with a 9.4
Tesla magnet (1H: 400 MHz; 13C: 100 MHz); GC 2010 Plus
and MSTQ8030 (Shimadzu); MDS-Sciex API 2000 LC-ESI-
MS/MS (Applied Biosystems, Foster, CA, USA); HPLC
(Shimadzu) coupled to an Esquire 6000 ion trap (IT) mass
spectrometer (Bruker) (LC-IT-MS/MS); and LC (Agilent,
Barueri, SP, Brazil) coupled to an automatic cartridge
exchanger Bruker/Spark Prospekt 2 SPE unit (Bruker
BioSpin, Rheinstetten, Germany) and a Bruker Daltonik
NMR 600 MHz spectrometer with a 14.1 Tesla magnet
(LC-SPE-NMR).
Chemical modification of the phenolic compounds
from ginger could improve their pharmacological prop-
erties and potential therapeutic applications. Organic
synthesis is challenging, but the microorganisms or their
enzymatic systems may be powerful tools for accom-
ꢀ
ꢀ
plishing desired chemical transformations (Garcıa-Pajon
et al. 2003). The biotransformation of [6]-shogaol by
Aspergillus niger (Takahashi et al. 1993; Lee 1995; Lee
and Lee 1995; Jo et al. 2016) and Schizosaccharomyces
pombe (Choi et al. 2017) produced new lead com-
pounds, such as [6]-paradol, which had significant
pharmacological activities and higher bioavailability than
[6]-shogaol (Jo et al. 2016; Choi et al. 2017).
In this work, we increased the chemical diversity of
ginger metabolites by using the pathogenic fungus
Colletotrichum gloeosporioides to transform ginger con-
stituents (zingerone, [6]-shogaol, [6]-gingerol, and
methyl-[6]-gingerol). Colletotrichum gloeosporioides is
capable of oxidoreductase activity, promoting regiose-
lective hydroxylations and diastereo- and enantioselec-
tive reductions on different substrates (Miyazawa et al.
Solvents for chromatographic procedures (ethanol,
n-hexane, ethyl acetate (EtOAc), and methanol
(MeOH)) were from Vetec (Duque de Caxias, RJ, Brazil).
HPLC grade solvents (MeOH, acetonitrile (ACN), and
water) were obtained from Merck (Darmstadt, HE,
Germany). Deuterated solvents (chloroform (CDCl3),
methanol (CD3OD), and tetramethylsilane (TMS)) were
acquired from and Sigma-Aldrich. Silica gel 60 (SiO2,
70–230 mesh) and thin-layer chromatography (TLC) on
pre-coated aluminium silica 60 F254 were from Merck.
TLC was used to monitor compounds from chroma-
tography procedures and from synthesis reactions by
UV254/366 and reaction with sulphuric vanillin solution.
ꢀ
ꢀ
1995; Miyazawa and Wada 2000; Garcıa-Pajon et al.
2003). To investigate the fungal biotransformation of
ginger metabolites, compounds with and without deu-
terium labelling were prepared as substrates for C.
gloeosporioides, and fungal metabolites were analyzed
using GC-MS, LC-MS/MS, and LC-SPE-NMR.
Materials and methods
General materials
The fungus C. gloeosporioides (Penz.) Penz. & Sacc. was
furnished by the Laboratory of Physiology and
Phytopathological Biochemistry, Luiz de Queiroz College
~
of Agriculture, University of Sao Paulo (USP, Piracicaba,
Microorganism pre-inoculation with the substrate
SP, Brazil). Potato dextrose agar (PDA) medium was
obtained from Neogen Corporation (Lansing, MI, USA),
Cultures of C. gloeosporioides were maintained on
potato dextrose agar (PDA) plates at 25 ꢀC. After
and
zingerone
(CAS
122-48-5)
and
lithium

BIOCATALYSIS AND BIOTRANSFORMATION
3
incubation for 7 days, spores and mycelia were sus-
pended in sterile water (2 mL). To evaluate substrate
toxicity, experiments in test tubes and enzyme-linked
immunosorbent assay (ELISA) 96-well plates were per-
formed. Serial dilutions using a stock solution of
1600 mg mLꢁ1 led to concentrations of 0.1–15 mg
mLꢁ1 in the test tubes. In the ELISA plate experiments,
20 mL of inoculum (spore and mycelium suspension)
was mixed with 80 mL of sterile culture medium (PDA)
contained fungi culture in each well. Then 100 mL of
individual substrates dissolved in sterile water and
DMSO (zingerone, [6]-shogaol, [6]-gingerol, and
methyl-[6]-gingerol) were added to final concentra-
tions of 0.781–400 mg mLꢁ1. The positive control was
the antifungal compound actidione and the negative
control was sterile PDA medium without substrate.
After incubation of both test tubes and plates for
15 days at 30 ꢀC, toxicity was visually evaluated by
observing mycelium growth.
NMR experiments as a zingerone dimer, 4,40-(6,60-dihy-
droxy-5,50-dimethoxy-[1,10-biphenyl]-3,30-diyl)
bis
(butan-2-one)] (1), (Slavova-Kazakova et al. 2015). ESI-
MS: [M þ Na]þ m/z 409.1648, M of m/z (C22H26O6,
1
386.1729 calcd.); H NMR (CDCl3) dH ppm 2.16 (s, 6H),
2.78 (d, 4H), 2.88 (d, 4H), 3.92 (s, 6H), 6.75 (d,
J ¼ 4.0 Hz, 2H), 6.73 (d, J ¼ 4.0 Hz, 2H).
Optimized conditions with substrates: zingerone,
zingerone-D5, [6]-gingerol, [6]-gingerol-D3,
methyl-[6]-gingerol and [6]-shogaol
Three C. gloeosporioides mycelial plugs (0.5 cm diam-
eter), cut from the growing edge of a PDA plate, were
transferred under sterile conditions to Erlenmeyer
flasks (250 mL) containing 50 mL of sterile liquid PD.
After 48 h of fermentation (30 ꢀC, 120 rpm), 0.5 mL ali-
quots of substrate solutions (100 mg mLꢁ1 in DMSO)
were added and the cultures were incubated for
10 days (30 ꢀC, 120 rpm). A control without substrate
addition (control A) and control without fungal inocu-
lation (control B) were prepared in the same condi-
tions. All experiments were performed in triplicate.
After the incubation period, the culture media con-
taining mycelia were filtered. The mycelia were
extracted with MeOH for 4 days and agitated with an
ultrasound probe for 30 min. The filtrate was extracted
with EtOAc (3 ꢂ 20 mL). Solvent removal by rotary
evaporation under reduced pressure (40 ꢀC) yielded an
organic extract of EtOAc, a methanolic extract (MM),
and an aqueous extract. The extraction process for the
controls was performed in the same manner. Fungal
metabolites were identified in the EtOAc extracts of
samples by LC-MS/MS, LC-SPE-NMR, and GC-MS.
Large-scale biotransformation of zingerone and
purification of products
Two C. gloeosporioides mycelial plugs (0.5 cm diam-
eter), cut from the growing edge of a PDA plate, were
added to 60 mL test tubes containing 15 mL of sterile
liquid potato dextrose (PD). After 7 days of fermenta-
tion (30 ꢀC, 120 rpm), 1.0 mL of this active mycelium
suspension was transferred to 30 mL test tubes con-
taining 8 mL of sterile liquid potato dextrose and 1 mL
of zingerone dissolved in sterile water and DMSO (final
concentrations: 1.0; 5.0; 10.0; 15.0 mg mLꢁ1). After
15 days of incubation at room temperature, mycelium
growth was only observed in tubes containing 1.0 mg
mLꢁ1 zingerone. The control without substrate (con-
trol A) and control without fungal inoculation but with
the substrate (control B) were prepared in the same
conditions. All controls and experiments were per-
formed in quintuplicate.
After the incubation period, culture media contain-
ing mycelia were filtered. The combined filtrate
obtained from test tubes with 1.0 mg mLꢁ1 of zinger-
one was extracted with EtOAc (3 ꢂ 20 mL), affording
extract (A) (9 mg). The tubes with 5–15 mg mLꢁ1 of
zingerone were combined and extracted with EtOAc
(3 ꢂ 60 mL), affording extract (B) (960 mg). The solvent
from extracts A and B was removed by rotary evapor-
ation under reduced pressure (40 ꢀC). The extract (B)
Experiments for metabolite profiling
The EtOAc extract and control A, control B, were
diluted in 10% MeOH (1 mg mLꢁ1 in water) and fil-
tered (Millipore, 0.20 mm).
For Methods I, II, and III, an Eclipse XDB-C18
Phenomenex (5 lm particle size; 150 ꢂ 4.6 mm)
(Agilent) column connected to a C18 (5 lm; 4 ꢂ 30 mm)
~
(Allcrom, Sao Paulo, SP, Brazil) was used. The flow rate
for all methods was 1.0 mL minꢁ1. Unless otherwise
noted, peak detection was monitored at 220 nm.
Method I: The mobile phase was composed of H2O
(A) and ACN or MeOH (B). The method started with a
gradient of 10–37% (B) over 3 min, followed by 37%
(B) for 7 min (isocratic), a linear gradient of 37–55% (B)
over 2 min, a linear gradient of 55–100% (B) over
10 min (%D ACN ¼ 4.5), and 100% (B) for 12 min. The
was fractionated on
a
silica gel 60 column
(2.5 ꢂ 11 cm; n-hexane–EtOAc 7:3 (1000 mL, isocratic)),
affording 17 fractions. The fourth fraction (N-4)
(4.0 mg) was identified by high-resolution MS and

4
R. M. D. DE ÁVILA ET AL.
column was cleaned for 20 min using 100% (B), and a
conditioning cycle time of 30 min brought the column
back to the initial condition (10% (B)).
Method II: The mobile phase was composed of H2O
(A) and MeOH or ACN (B). The method started with
5 min of 10% (B), followed by a linear gradient of
10–100% (B) over 20 min (%D MeOH ¼ 4.5), and
20 min of 100% (B). Column cleaning and a condition-
ing cycle time of 20 min returned the column to the
initial condition (10% (B)).
LC-SPE-NMR
The LC-SPE-NMR system contained the following
instruments: LC system 1200 series, GmbH, G1311A
quaternary pump, G1322A degasser, G1315D, photo-
diode array detector, G1329A autosampler (Agilent,
Barueri, SP, Brazil); automatic cartridge exchanger
Bruker/Spark Prospekt 2 SPE unit (Bruker BioSpin,
Rheinstetten, Germany), containing SPE cartridges
(HySphere Resin GP, 10 ꢂ 2.0 mm, 10 mm spherical pol-
ydivinylbenzene stationary phase); Bruker Daltonik
NMR 600 MHz spectrometer with a 14.1 Tesla magnet,
Method III: The mobile phase consisted of H2O (A)
and ACN (B) with 0.1% v/v formic acid in both sol-
13
1Hf C, 15Ng TCI triple resonance cryogenically-cooled
probe head. The software used for data acquisition
was HYSTAR v. 3.2 (Bruker BioSpin GmbH).
vents.
A
linear gradient increasing solvent (B)
10–100% (%D MeOH ¼ 4.5), followed by 20 min of
100% (B) was used. Column cleaning and a condition-
ing cycle time of 20 min returned the column to the
initial condition (10% (B)).
GC-MS
GC experiments were performed in electron impact
(EI) ionization mode (70 eV). The split mode was used.
An aliquot (1 lL) of the sample was injected onto the
GC-MS outfitted with an Optima-5 column
(30 ꢂ 0.25 mm and film 0.25 lm; Shimadzu) starting at
70 ꢀC. Theꢁm1 ethod used was as folloꢁw1s: 70 ꢀC (6 min),
LC-ESI-MS/MS
Selected reaction monitoring (SRM) experiments were
carried out in positive [M þ H]þ and negative [M ꢁ H]ꢁ
modes using an HPLC (Agilent) coupled to a API2000
spectrometer containing a triple quadrupole and an
15 ꢀC min
up to 160 ꢀC,ꢁ51 ꢀC min
up to 200 ꢀC,
200 ꢀC for 2 min, 20 ꢀC min up to 260 ꢀC; 260 ꢀC for
10 min. The flow rate was 1.2 mL minꢁ1. The data
electrospray
ionization
source
(ESI)
(Applied
R
were analyzed using GCMS Real-Time AnalysisV
Biosystems). Standard samples (10 mg mLꢁ1) of sub-
strates and product metabolites (described above)
were prepared in 7:3 MeOH:H2O. The standard sam-
ples were used to optimize parameters for direct injec-
tion. Declustering potential (DP), collision exit
potential (CXP), collision energy (CE), and entrance
potential (EP) were optimized for each analyte. The
parameters used were as follows: DP (V), ꢁ35, 3; CXP
(V), ꢁ3, 45; CE (V), ꢁ50, 25; EP (V), ꢁ10, input tem-
perature of 400 ꢀC.
(Shimadzu) and the National Institute of Standards &
Technology (NIST) Mass Spectral Library.
Results
Optimization of conditions for substrate
biotransformation
The maximum concentration of substrate with visible
C. gloeosporioides mycelial growth after 15 days of
incubation was 1 mg mL21. To ensure the biotrans-
formation of substrates, the experiments were subse-
quently carried out using 100 mg mL21 of the
substrate, a concentration that did not visibly cause
toxicity to the microorganism. Afterwards, the filtrates
of culture media containing mycelia were extracted
with EtOAc, and the EtOAc extracts were analyzed for
LC-IT-MS/MS
Experiments were performed on an HPLC (Shimadzu)
coupled to an Esquire 6000 IT-MS (Bruker) operating
in positive [M þ H]þ and negative [M ꢁ H]ꢁ ESI mode.
The parameters used were as follows: nebulizer, 30 psi;
dry gas flow, 8.0 L minꢁ1; dry heater temperature,
325 ꢀC; capillary voltage, 4.5 kV; collision cell energy,
0.5 V in negative mode and 1.0 V in positive mode;
smart parameter settings, m/z 300 for negative mode
and m/z 277 for positive mode; full-MS scan range, m/
z 100–600; and flow (splitter) 0.1 mL minꢁ1. Spectra
were obtained in auto MS/MS mode with a CE of
20–40 eV for all m/z in the analyzed range. Data
Analysis 4.0 software was used for spectral analysis.
the
metabolite
profile
of
each
substrate
biotransformation.
Biotransformation of zingerone to the zingerone
dimer and its isolation
Large-scale biotransformation of zingerone leads to
EtOAc extract (B) (960 mg), which, after chromato-
graphic purification, yielded the zingerone dimer (1)
(4.0 mg). The structure of 1 was elucidated using 1H

BIOCATALYSIS AND BIOTRANSFORMATION
5
NMR, high-resolution MS (Bruker Daltonics, Micromass
TOF–QII-ESI-TOF), and comparison with literature
(Slavova-Kazakova et al. 2015). The zingerone dimer
(1) had a molecular ion m/z 409.1648 ([M þ Na]þ)
using positive mode and an m/z 385.1700 ([M ꢁ Na]ꢁ)
using the negative mode, indicating the molecular for-
mula C22H26O6 and an M of m/z (C22H26O6, 386.1729
calcd.). Direct injection in negative mode showed an
[M ꢁ H]ꢁ ion at m/z 385 and the fragment ions m/z
2.0 Hz, 1H), dH 6.70 (d, J ¼ 8.0 Hz, 1H), and dH 6.79 (d,
J ¼ 2.0 Hz, 1H), which are typical of a 1,2,4-trisubsti-
tuted aromatic ring. The presence of a methoxyl
group and 2 methylene groups are shown by the sing-
let at dH 3.83 (s, 3H) and 2 triplets at dH 2.73 (t, 2H)
and dH 3.70 (t, 2H), respectively.
Compound (3) is a biphenol dimer. The structure
was confirmed by the [M ꢁ H]ꢁ ion at m/z 333 with
fragment ions m/z 287, 215, 187, 171 and 143. In the
1H NMR, the signal at dH 3.89 (s, 6H) was attributed to
the 2 methoxyl groups, and the 2 triplets at dH 2.75 (t,
4H) and dH 2.77 (t, 4H) were attributed to methylene
groups. The aromatic hydrogens at dH 6.75 (d;
J ¼ 2.0 Hz, 2H) and dH 6.73 (d; J ¼ 2.0 Hz, 2H) indicate
the symmetry of this molecule (Pulvirenti et al. 2017).
SRM analysis (LC-ESI-MS/MS) was used with transi-
tions of m/z 167 to 93 and m/z 333 to 215 to evaluate
the time course of the biotransformation of [6]-gin-
gerol to compounds 2 and 3 (days 1–7 and 10)
(Figure 2(B)). Results were compared to controls (A
and B). The 4-(2-hydroxyethyl)-2-methoxyphenol (2)
peak was higher in intensity on days 1 and 5, but the
biphenol dimer (3) was highest on day 10.
1
370 and m/z 327. The H NMR spectrum revealed sig-
nals for aromatic hydrogens at dH 6.75 (d; J ¼ 4.0 Hz)
and dH 6.73 (d; J ¼ 4.0 Hz), indicating a meta relation-
ship of hydrogens from 2 tetrasubstituted aromatic
rings. The symmetry of the molecule generates over-
lapped signals since some protons are chemically and
magnetically equivalent. Likewise, the signal at dH 3.92
(s, 6H) was attributed to the 2 methoxyl groups bound
to the aromatic rings. Another singlet observed at dH
2.16 (s, 6H) was characteristic of a methyl ketone
group. Two triplets at dH 2.78 (t, 4H) and dH 2.88 (t,
4H) were attributed to methylene groups, confirming
the structure of the zingerone dimer (1) (Figure 1).
To investigate the possible reactions involved in
the biotransformation of [6]-gingerol, and to eliminate
the possibility that compound 2 was a derivative of
tyrosol or another fungal metabolite (Bungihan et al.
2013), we carried out the biotransformation of deuter-
ated [6]-gingerol ([6]-gingerol-D3; 54%), administering
Metabolite profiling of [6]-gingerol, [6]-gingerol-
D3, zingerone-D5 and methyl-[6]-gingerol
biotransformation by C. gloeosporioides
The chromatographic analysis of EtOAc extracts
revealed complete substrate degradation by day 10,
observed by the lack of the [6]-gingerol peak at (tR) ¼
21.5 min (Figure 2(A)). The product metabolites were
identified as 4-(2-hydroxyethyl)-2-methoxyphenol (2)
((tR) ¼ 12.5 min) and 5,50-bis(2-hydroxyethyl)-3,30-dime-
thoxy-[1,10-biphenyl]-2,20-diol (3) ((tR) ¼ 16.3 min).
4-(2-hydroxyethyl)-2-methoxyphenol (2) was identi-
fied by the [M ꢁ H]ꢁ ion at m/z 167 and by character-
the labelled compound as
gal culture.
a substrate in fun-
We also used penta-deuterated zingerone (zinger-
one-D5; 66.3%) (100 mg mL21) as a model compound
in biotransformation reactions with C. gloeosporioides.
The time course of the biotransformation was moni-
tored by analyzing EtOAc extracts from days 1, 3, 6, 8
and 10. The chromatogram (Method I) of the EtOAc
extracts showed zingerone degradation on day 6 and
1
istic signals in the H NMR (600 MHz) (Christophoridou
and Dais 2009), including dH 6.65 (dd, J ¼ 8.0 and
Figure 1. Zingerone dimer (1), the product of the biotransformation of zingerone by C. gloeosporioides. Phenolic oxidative cou-
plings of zingerone are proposed based on a hydrogen-abstraction of phenol. The zingerone dimer is the product of ortho–ortho
coupling by enolization, which restores aromaticity to rings.

6
R. M. D. DE ÁVILA ET AL.
an increase of zingerone dimer (1) (Figure S5,
Supplementary Information).
The zingerone biotransformation metabolite profile
was also analyzed by LC-MS (Method III, LC-IT-MS/MS).
Figure 3 shows the ion chromatograms of the EtOAc
extract for the time course experiment. Biphenol for-
mation was observed ([M ꢁ H]ꢁ ion with m/z 385).
Fragmentation of the product metabolite (day 10,
Figure 2. (A) HPLC chromatogram of EtOAc extract of the bioconversion of [6]-gingerol (100 mg mLꢁ1) by C. gloeosporioides after
10 days. Control (A): without substrate addition; Control (B): substrate without fungal inoculation ((tR) ¼ 21.5 min). EtOAc extract:
consumption of [6]-gingerol by the fungus shows a lack of the substrate peak at (tR) ¼ 21.5 min. HPLC conditions: Method II with
water (A):MeOH (B); (B) SRM analysis (LC-ESI-MS/MS) with transitions of m/z 167 to 93 and m/z 333 to 215, product metabolites
(2) and (3), respectively, during time course of biotransformation (days 1–7 and 10).
Figure 3. Representative LC-MS extracted ion chromatogram for EtOAc extract obtained from culture medium of C. gloeosporioides
with 100 mg mL21 of zingerone and its metabolites. Method III was used for the experiment (LC-IT-MS/MS).

BIOCATALYSIS AND BIOTRANSFORMATION
7
peak at (tR) ¼ 10.0 min) produced the fragment ions
m/z 370.03 and 327.01, characteristic of compound 1.
The bioconversion of zingerone-D5 led to distinct
deuterium labelling. The [M ꢁ H]ꢁ ion at m/z 389.12,
corresponding to the penta-deuterated zingerone
dimer, confirmed that zingerone was recognized by C.
gloeosporioides enzymes and was incorporated into its
metabolic pathways (Figure S9, Supplementary
Information).
After confirmation that labelled zingerone was
transformed by C. gloeosporioides into the labelled
dimer, we performed large-scale biotransformations of
[6]-gingerol and [6]-gingerol-D3 for 10 days, which
afforded 15.0 mg of EtOAc extract.
The transformation of the carbon chain of [6]-gin-
gerol-D3 was verified by identification of the biphenol
dimer-D3 in the EtOAc extract using LC-IT-MS/MS
(Method III). Based on the fragmentation pattern
(Figure S14, Supplementary Information), it is possible
that the biotransformation of [6]-gingerol could occur
by a fungal enzyme-catalyzed Baeyer–Villiger reaction
followed by hydrolysis, affording 4-(2-hydroxyethyl)-2-
methoxyphenol (2) (Figure 4).
Methyl-[6]-gingerol, a metabolite from the ginger
root (Li et al. 2009), was obtained by chemical derivati-
zation of [6]-gingerol, and the derivative was supplied
to C. gloeosporioides cultures with the same optimized
conditions. The EtOAc extract obtained on day 5 was
analyzed using Method II (water (A):MeOH (B)), GC-MS
and LC-IT-MS/MS (Method III) (Figures S19–S25 and
Table S1, Supplementary Information). The metabolic
products 3-methoxy-4-hydroxyphenylethanol (2) and
3,4-dimethoxyphenylethanol (4) were observed. The
production of compound 2 could be related to a
remote possibility that some [6]-gingerol remained
after purification of methyl-[6]-gingerol, or that a
demethylation reaction occurred. Compound 4 was
assigned based on the GC-MS observation of the m/z
182 ion and its fragments (m/z 151, 135, 107, 91, 77,
and 51), as well as 1H NMR data and a comparison
with literature (Shahane et al. 2008). This indicates
methyl-[6]-gingerol follows the same fungal degrad-
ation pathway as [6]-gingerol (Figure 4).
The chromatogram peaks observed between (tR) ¼
5 and 7 min, which were absent in control (B), were
analyzed in positive mode ((þ)-ESI-MS/MS). The
[M þ H]þ ions m/z 325 and 327 and the fragment ions
(m/z 307, 289, 191, and 133; m/z 309, 291, 273, 221,
191, 177, and 151, respectively) suggested the product
metabolites M1 (m/z 325), M2 (m/z 327), and M3 (m/z
327), products of hydroxylation and reduction reac-
tions. Compound M1 eluted at (tR) ¼ 6.4 min. M2 and
M3 were identified in peaks with different retention
times: (tR) ¼ 6.0 and 6.3 min and (tR) ¼ 5.8 and
Figure 4. Proposed fungal degradation pathway of [6]-gingerol-D3. The [M ꢁ H]ꢁ with m/z 503 indicated a phenol trimer-D3 prod-
uct of phenolic oxidative coupling of 4-(2-hydroxyethyl)-2-methoxyphenol (2); the ions at m/z 169 and m/z 337 are related to the
deuterated product metabolites (2) and (3) (Figure 2(B), m/z 167 and m/z 333, respectively).

8
R. M. D. DE ÁVILA ET AL.
Figure 5. Metabolic products of the bioconversion of methyl-[6]-gingerol.
5.9 min, respectively, which indicates that both may
exist as diastereoisomer pairs (Figure 5).
137 suggested the presence of [6]-gingerdiol (6), a
natural constituent of Zingiber officinale roots (Kikuzaki
et al. 1992; Jolad et al. 2004). [6]-gingerdiol (6) could
be a product of [6]-shogaol carbonyl reduction, a reac-
tion which has been observed in C. gloeosporioides
Metabolite profiling of [6]-shogaol
biotransformation by C. gloeosporioides
ꢀ
ꢀ
cultures with several other substrates (Garcıa-Pajon
et al. 2003). Additionally, in a previous study of
Aspergillus niger cultures supplemented with [6]-sho-
gaol, product metabolites were mostly formed
through double bond and ketone reduction, and
hydroxylation (Jo et al. 2016).
The day 5 EtOAc extract obtained after [6]-shogaol
incubation with the fungus revealed substrate con-
sumption by C. gloeosporioides, although higher prod-
uct metabolite intensities were observed after 10 days.
Five compounds were identified by MS as product
metabolites from [6]-shogaol biodegradation: 3-
methoxy-4-hydroxyphenylethanol (2), 2-octenoic acid,
2-(4-hydroxy-3-methoxyphenyl)ethyl ester (5), [6]-gin-
gerdiol (6), tyrosol (7), and octanoic acid, 2-(4-hydroxy-
3-methoxyphenyl)ethyl ester (8).
GC-MS analysis of the day 5 EtOAc extract allowed
the identification of compounds 2, 5, 6, and 7, which
were confirmed by comparing to literature data
(Kikuzaki et al. 1992; Jolad et al. 2004; Christophoridou
and Dais 2009). Compounds 6 and 8 were also found
on day 10. The characteristic ions m/z 168, 137 and
122 belong to 4-(2-hydroxyethyl)-2-methoxyphenol (2),
which was also identified as the product metabolite
from [6]-gingerol and methyl-[6]-gingerol biodegrad-
ation, as previously discussed. The m/z 292 ion,
together with the m/z 137 base peak, was suggestive
of the ester 5, an intermediate metabolite in a
Baeyer–Villiger reaction. The Baeyer–Villiger reaction
could be catalyzed by an oxidase enzyme, such as a
peroxidase or a laccase (Figure 6), reinforcing our pre-
vious proposed reaction pathway for 4-(2-hydrox-
yethyl)-2-methoxyphenol (2) (Figure 4).
The parent peak ion at m/z 294 with a base peak
m/z 137 could be hydroxylation of [6]-shogaol to give
[6]-gingerol, as fragment data match the literature val-
ues. However, the retention time observed for com-
pound 8 ((tR) ¼ 28.5 min) was quite different from the
standard [6]-gingerol ((tR) ¼ 27.8 min, Figure S27)
using the same experimental conditions. In addition,
the product ions expected from the thermal degrad-
ation of [6]-gingerol were absent in the GC-MS chro-
matogram. Thus, the m/z 294 ion was attributed to
the formation of ester 8, which could be the product
of the double bond reduction of ester 5 (Gan et al.
2016). Tyrosol (7), a molecule previously isolated from
C. gloeosporioides (Bungihan et al. 2013), was identified
by the ion with m/z 138 and a base peak at m/z 107
(Figure 7).
The metabolite profile of [6]-shogaol biotransform-
ation was also analysed by LC-MS (Method III, LC-IT-
MS/MS) on days 5 and 10 (Figure 8). Product metabo-
lites were identified, especially with retention times
between 5 and 10 minutes, that were not observed in
the control samples.
The identification of molecular and fragmentation
ions m/z 296, 278, 260, 207, 189, 175, 163, 150 and
The [M ꢁ H]ꢁ ion at m/z 295 ((tR) ¼ 7.2 min) with a
fragment ion at m/z 280 is related to [6]-gingerdiol (6)

BIOCATALYSIS AND BIOTRANSFORMATION
9
Figure 6. Proposed degradation pathway of [6]-shogaol by C. gloeosporioides. The ester (5) is a possible intermediate metabolite
in a Baeyer–Villiger reaction, leading to the formation of 4-(2-hydroxyethyl)-2-methoxyphenol (2).
Figure 7. Metabolic products from the biotransformation of ginger constituents (zingerone, [6]-shogaol, [6]-gingerol, and methyl-
[6]-gingerol) by C. gloeosporioides.
(Tao et al. 2009), and the [M ꢁ H]ꢁ ion at m/z 291((tR)
Discussion
¼ 9.3 min) with fragments at m/z 276, 156 and 123 is
related to the proposed ester 5. Compounds 5 and 6
were identified in both day 5 and day 10 extracts. The
[M ꢁ H]ꢁ ion at m/z 291 could also be [6]-gingerdione,
a product of oxidoreductase activity. However, the
fragment ions in the spectra were not identical to
fragment ions from confirmed [6]-gingerdione using
the same conditions (ESI-MS/MS) (Tao et al. 2009).
Many members of the Colletotrichum genus are plant
pathogens. Species of this genus have been widely
investigated in order to understand how their meta-
ꢀ
ꢀ
bolic cause crop diseases (Garcıa-Pajon et al. 2003).
However, the biotransformation of ginger metabolites
by C. gloeosporioides has not been previously
investigated.

10
R. M. D. DE ÁVILA ET AL.
Figure 8. Chromatogram of EtOAc after 5 and 10 days of [6]-shogaol (100 mg mL21) biodegradation by C. gloeosporioides. (A)
EtOAc extract analysis after 5 days; (B) EtOAc extract analysis after 10 days; (C) same as (B) but without C. gloeosporioides; HPLC
conditions: Method III, using water (A):ACN (B) with 0.1% v/v formic acid in both solvents (LC-IT-MS/MS, Full scan in nega-
tive mode).
The fungal biotransformation of substrates from
ginger (zingerone, [6]-shogaol, [6]-gingerol, and
methyl-[6]-gingerol) afforded 11 metabolic products of
fungal bioconversion (Figure 7). Zingerone and [6]-gin-
gerol were transformed into the zingerone dimer (1)
degrade dyes in industrial effluents (Wang et al. 2016).
A laccase from C. gloeosporioides also metabolises epi-
catechin from avocado (Guetsky et al. 2005). The
biphenol dimer (3) could be also a product of phen-
olic oxidative coupling catalyzed by oxidase enzymes
(Figure 4), probably initiated by the hydrogen-abstrac-
tion of compound 2, which is a product metabolite
identified from [6]-gingerol biodegradation (Jeon
et al. 2012).
and
5,50-bis(2-hydroxyethyl)-3,30-dimethoxy-[1,10-
biphenyl]-2,20-diol (3), respectively. Biphenols are use-
ful compounds for organic synthesis and have many
industrial, pharmaceutical, and agricultural applications
(Bonrath et al. 2007; Engelmann et al. 2015). The zin-
gerone dimer was synthesized previously and was
demonstrated to be a biphenol with antioxidant activ-
ity (Marchiani et al. 2013; Slavova-Kazakova et al.
2015). The biphenol dimer (3) reduces the catalytic
activity of yeast a-glucosidase, an enzyme related to
diabetic diseases (Pulvirenti et al. 2017).
Compound
2
was obtained when supplying
methyl-[6]-gingerol and [6]-shogaol as substrates for C.
gloeosporioides. Another intermediate of the proposed
reaction pathway for [6]-shogaol biodegradation was
the ester (5) (Figure 6), the presence of which sug-
gests a fungal enzyme-catalysed Baeyer–Villiger reac-
tion, followed by hydrolysis.
Oxidase enzymes (peroxidases and laccases) likely
formed the zingerone dimer by phenolic oxidative
coupling of the products of free radical reactions
(Jeon et al. 2012). Biphenols with C–C couplings are
mostly produced by fungal laccase enzymes, which
are copper-containing oxidoreductases with redox
potential and have applications in biotechnology
(Hollmann et al. 2011; Jeon et al. 2012; Abdel-Mohsen
et al. 2014; Engelmann et al. 2015). Previously, a lac-
case from Colletotrichum lagenarium was shown to
Conclusions
In conclusion, labelling the pungent constituents of
ginger with deuterium allowed verification of the fun-
gal biotransformation of the metabolites by GC-MS,
LC-MS/MS, and LC-SPE-NMR. The bioconversion of [6]-
gingerol and zingerone led to biphenol dimers, sug-
gesting that C. gloeosporioides metabolises these sub-
strates using an oxidase enzyme, possibly a laccase.

BIOCATALYSIS AND BIOTRANSFORMATION
11
ginger in osteoarthritis patients:
randomized placebo-controlled trials. Osteoarthritis Cartil.
23(1):13–21.
A meta-analysis of
For the 4-(2-hydroxyethyl)-2-methoxyphenol metabol-
ite was proposed a degradation pathway by C. gloeo-
sporioides involving Baeyer–Villiger reaction and
subsequent hydrolysis. Colletotrichum gloeosporioides
enzymes mostly performed reduction and hydroxyl-
ation reactions on methyl-[6]-gingerol and [6]-shogaol.
We report for the first time the ability of C. gloeospor-
ioides to perform phenolic oxidative coupling reac-
tions. Our results demonstrate the utility of fungi in
transforming complex metabolites into biphenols,
which have broad industrial interest.
Bhattarai S, Tran VH, Duke CCJ. 2007. Stability of [6]-gingerol
and [6]-shogaol in simulated gastric and intestinal fluids. J
Pharm Biomed Anal. 45(4):648–653.
Bonrath W, Eggersdorfer M, Netscher T. 2007. Catalysis in
the industrial preparation of vitamins and nutraceuticals.
Catal Today. 121(1–2):45–57.
Bungihan ME, Tan MA, Takayama H, Dela Cruz TEE, Nonato
MG. 2013. A new macrolide isolated from the endophytic
fungus Colletotrichum sp. Philippine Sci Lett. 6:57–73.
Choi JG, Kim SY, Jeong M, Oh MS. 2018.
Pharmacotherapeutic potential of ginger and its com-
pounds in age-related neurological disorders. Pharmacol
Ther. 182:56–69.
Disclosure statement
No potential conflict of interest was reported by
the author(s).
Choi JW, Park HY, Oh MS, Yoo HH, Lee SH, Ha SK. 2017.
Neuroprotective effect of [6]-paradol enriched ginger
extract by fermentation using Schizosaccharomyces
pombe. J Funct Foods. 31:304–310.
Christophoridou S, Dais P. 2009. Detection and quantification
of phenolic compounds in olive oil by high resolution 1H
nuclear magnetic resonance spectroscopy. Anal Chim
Acta. 633(2):283–292.
Citronberg J, Bostick R, Ahearn T, Turgeon DK, Mack TR,
Djuric Z, Ananda S, Brenner DE, Zick SM. 2013. Effects of
ginger supplementation on cell-cycle biomarkers in the
normal-appearing colonic mucosa of patients at increased
risk for colorectal cancer: Results from a pilot, randomized,
and controlled trial. Cancer Prev Res. 6(4):271–281.
Da Silva JA, Becceneri AB, Mutti HS, Martin ACBM, Silva M,
Fernandes JB, Vieira PC, Cominetti MR. 2012. Purification
and differential biological effects of ginger-derived sub-
stances on normal and tumor cell lines. J Chromatogr B.
903:157–162.
Funding
~
The present work was financially supported by the Sao
Paulo Research Foundation (FAPESP) (Grant # 14/50249-8)
and the Coordination for the Improvement of Higher
Education Personnel (CAPES, Finance Code 001).
ORCID
Leonardo Toffano
http://orcid.org/0000-0002-7591-0530
http://orcid.org/0000-0002-
~
Joao Batista Fernandes
2924-3327
ꢀ
Maria Fatima das Grac¸as Fernandes da Silva
d.org/0000-0002-7081-817X
http://orci-
Lorena Ramos Freitas de Sousa
0002-2506-8365
http://orcid.org/0000-
Engelmann C, Illner S, Kragl U. 2015. Laccase initiated C–C
couplings: Various techniques for reaction monitoring.
Process Biochem. 50(10):1591–1599.
Paulo Cezar Vieira
http://orcid.org/0000-0003-4207-6217
ꢀ
Fuzer AM, Martin ACBM, Becceneri AB, da Silva JA, Vieira PC,
Cominetti MR. 2019. [10]-Gingerol affects multiple meta-
static processes and induces apoptosis in MDAMB- 231
Breast Tumor Cells. Anticancer Agents Med Chem. 19(5):
645–654.
References
Abdel-Mohsen HT, Conrad J, Beifuss U. 2014. Laccase-cata-
lyzed synthesis of catechol thioethers by reaction of cate-
chols with thiols using air as an oxidant. Green Chem.
16(1):90–95.
Gan Z, Liang Z, Chen X, Wen X, Wang Y, Li M, Ni Y. 2016.
Separation and preparation of 6-gingerol from molecular
distillation residue of Yunnan ginger rhizomes by high-
speed counter-current chromatography and the antioxi-
dant activity of ginger oils in vitro. J Chromatogr B Analyt
Technol Biomed Life Sci. 1011:99–107.
Ahmad B, Rehman MU, Amin I, Arif A, Rasool S, Bhat SA,
Afzal I, Hussain I, Sheikh B, Mir MR. 2015. A review on
pharmacological properties of zingerone (4-(4-Hydroxy-3-
methoxyphenyl)-2-butanone). Sci World J. 2015:816364.
Azimi P, Ghiasvand R, Feizi A, Hosseinzadeh J, Bahreynian M,
Hariri M, Khosravi-Boroujeni H. 2016. Effect of cinnamon,
cardamom, saffron and ginger consumption on blood
pressure and a marker of endothelial function in patients
with type 2 diabetes mellitus: A randomized controlled
clinical trial. Blood Press. 25(3):133–140.
Baliga MS, Shivashankara AR, Haniadka R, Palatty PL, Arora R,
Fayad R. 2013. Bioactive food as dietary interventions for
liver and gastrointestinal disease. vol. 11. Cambridge (MA):
Academic Press; p. 187–199.
Bartels EM, Folmer VN, Bliddal H, Altman RD, Juhl C, Tarp S,
Zhang W, Christensen R. 2015. Efficacy and safety of
ꢀ
ꢀ
ꢀ
ꢀ
Garcıa-Pajon CM, Hernandez-Galan R, Collado IG. 2003.
Biotransformations by Colletotrichum species. Tetrahedron:
Asymmetry. 14(10):1229–1239.
Guetsky R, Kobiler I, Wang X, Perlman N, Gollop N, Avila-
Quezada G, Hadar I, Prusky D. 2005. Metabolism of the fla-
vonoid epicatechin by laccase of Colletotrichum gloeospor-
ioides and its effect on pathogenicity on avocado fruits.
Phytopathology. 95(11):1341–1348.
Ho S, Chang K, Lin C. 2013. Anti-neuroinflammatory capacity
of fresh ginger is attributed mainly to 10-gingerol. Food
Chem. 141(3):3183–3191.

12
R. M. D. DE ÁVILA ET AL.
Hollmann F, Arends I, Buehler K, Schallmey A, Buhler B.
2011. Enzyme-mediated oxidations for the chemist. Green
Chem. 13(2):226–265.
Jeon JR, Baldrian P, Murugesan K, Chang YS. 2012. Laccase-
catalysed oxidations of naturally occurring phenols: From
in vivo biosynthetic pathways to green synthetic applica-
tions. Microb Biotechnol. 5(3):318–332.
Jo SK, Kim IS, Rehman SU, Ha SK, Park HY, Park YK, Yoo HH.
2016. Characterization of metabolites produced from the
biotransformation of [6]-shogaol formed by Aspergillus
niger. Eur Food Res Technol. 242(1):137–142.
Jolad SD, Lantz RC, Solyom AM, Chen GJ, Bates RB,
Timmermann BN. 2004. Fresh organically grown ginger
(Zingiber officinale): Composition and effects on LPS-
induced PGE2 production. Phytochemistry. 65(13):
1937–1954.
Kikuzaki H, Tsai SM, Nakatani N. 1992. Gingerdiol related
compounds from the rhizomes of Zingiber officinale.
Phytochemistry. 31(5):1783–1786.
Kou X, Wang X, Ji R, Liu L, Qiao Y, Lou Z, Ma C, Li S, Wang
H, Ho C-T. 2018. Ocurrence, biological activity and metab-
olism of [6]-shogaol. Food Funct. 9(3):1310–1327.
Kubra IR, Rao LJM. 2012. An impression on current develop-
ments in the technology, chemistry, and biological activ-
ities of ginger (Zingiber officinale Roscoe). Crit Rev Food
Sci Nutr. 52(8):651–688.
Kumar NV, Murthy PS, Manjunatha JR, Bettadaiah BK. 2014.
Synthesis and quorum sensing inhibitory activity of key
phenolic compounds of ginger and their derivatives. Food
Chem. 159:451–457.
Lee SS. 1995. Re-examination of [6]-shogaol biotransform-
ation by Aspergillus niger. Arch Pharm Res. 18(2):136–137.
Lee SS, Lee WY. 1995. Biotransformation of dehydroparadols
by Aspergillus niger. Arch Pharm Res. 18(6):458–461.
Li X, Ziyang L, Zhang H, Zhao L, Wu H, Zhang G, Wu Y, Chai
Y. 2009. Rapid LC-TOFMS separation and identification of
diarylheptanoids and gingerol-related compounds in dried
ginger. Chroma. 69(5–6):531–536.
by plant pathogenic fungus, Glomerella cingulate.
Phytochemistry. 40(4):1133–1137.
Miyazawa Y, Wada T. 2000. Biotransformation of gamma-ter-
pinene and (ꢁ)-alpha-phellandrene by the larvae of com-
mon cutworm (Spodoptera litura). J Agric Food Chem.
48(7):2893–2895.
Nile SH, Park SW. 2015. Chromatographic analysis, antioxi-
dant, anti-inflammatory, and xanthine oxidase inhibitory
activities of ginger extracts and its reference compounds.
Industr Crops Prod. 70:238–244.
Prasad S, Tyagi AK. 2015. Ginger and its constituents: Role in
prevention and treatment of gastrointestinal cancer.
Gastroenterol Res Pract. 2015:142979.
Pulvirenti L, Muccilli V, Cardullo N, Spatafora C, Tringali C.
2017. Chemoenzymatic synthesis and a-glucosidase inhibi-
tory activity of dimeric neolignans inspired by magnolol. J
Nat Prod. 80(5):1648–1657.
Semwal RB, Semwal DK, Combrinck S, Viljoen AM. 2015.
Gingerols and shogaols: Important nutraceutical principles
from ginger. Phytochemistry. 117:554–568.
Shahane S, Louafi F, Moreau J, Hurvois JP, Renaud JL, de
Weghe PV, Roisnel T. 2008. Synthesis of alkaloids of
Galipea officinalis by alkylation of an a-amino nitrile. Eur J
Org Chem. 2008(27):4622–4631.
Slavova-Kazakova AK, Angelova SE, Veprintsev TL, Denev P,
Fabbri D, Dettori MA, Kratchanova M, Naumov VV,
Trofimov AV, Vasil’ev RF, et al. 2015. Antioxidant potential
of curcumin-related compounds studied by chemilumines-
cence kinetics, chain-breaking efficiencies, scavenging
activity (ORAC) and DFT calculations. Beilstein J Org
Chem. 11:1398–3411.
Stoner GD. 2013. Ginger: Is it ready for prime time? Cancer
Prev Res. 6(4):257–262.
Takahashi H, Hashimoto T, Noma Y, Asakawa Y. 1993.
Biotransformation of [6]-gingerol and [6]-shogaol by
Aspergillus niger. Phytochemisty. 34(6):1497–1500.
Tamogami S, Agrawal GK, Rakwal R. 2016. Methyl jasmonate
elicits the biotransformation of geraniol stored as its glu-
cose conjugate into methyl geranate in Achyranthes biden-
tata plant. Plant Physiol Biochem. 109:166–170.
Tao Y, Li W, Liang W, Breemen RBV. 2009. Identification and
quantification of gingerols and related compounds in gin-
ger dietary supplements using high-performance liquid
chromatography-tandem mass spectrometry. J Agric Food
Chem. 57(21):10014–10021.
Marchiani A, Mammi S, Siligardi G, Hussain R, Tessari I,
Bubacco L, Delogu G, Fabbri D, Dettori MA, Sanna D,
et al. 2013. Small molecules interacting with a-synuclein:
Antiaggregating and cytoprotective properties. Amino
Acids. 45(2):327–338.,
ꢀ
Martin ACBM, Fuzer AM, Becceneri AB, da Silva JA, Tomasin
R, Denoyer D, Kim SH, McIntyre KA, Pearson HB, Yeo B,
et al. 2017. [10]-gingerol induces apoptosis and inhibits
metastatic dissemination of triple negative breast cancer
in vivo. Ocontarget. 8(42):72260–72271.
Villalvilla A, da Silva JA, da Largo R, Gualillo O, Vieira PC,
ꢀ
Herrero-Beaumont G, Gomez R. 2014. 6-Shogaol inhibits
chondrocytes’ innate immune responses and cathepsin-K
activity. Mol Nutr Food Res. 58(2):256–266. [
Mashhadi NS, Ghiasvand R, Askari G, Hariri M, Darvishi L,
Mofid MR. 2013. Anti-oxidative and anti-inflammatory
effects of ginger in health and physical activity: Review of
current evidence. Int J Prev Med. 4(1):S36–S42.
Wang B, Yan Y, Tian Y, Zhao W, Li Z, Gao J, Peng R, Yao Q.
2016. Heterologous expression and characterisation of a
laccase from Colletotrichum lagenarium and decolourisa-
Miyazawa M, Nankai H, Kameoka H. 1995. Biotransformations
of acyclic terpenoids, ( )-cis-nerolidol and nerylacetone,
tion of different synthetic dyes. World J Microbiol
Biotechnol. 32:1–9.