Biological Properties and Absolute Configuration of Flavanones From Calceolaria thyrsiflora Graham

Article abstract of DOI:10.3389/fphar.2020.01125

Flavanones (–)-(2S)-5,4’-dihydroxy-7-methoxyflavanone (1) and (–)-(2S)-5,3’,4’-trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of Chile. The absolute configuration of these compounds was resolved by optical rotation experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do structure-activity relationships with the biological assays studied. Biological tests revealed that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-resistant strain S. aureus MRSA 97-77 (MIC value of 50 μg/ml). In addition, flavanone 2 showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract. Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 ± 1.61 μM) but 4.6- and 17-fold lesser activity than etoposide and taxol.

Full text of DOI:10.3389/fphar.2020.01125

ORIGINAL RESEARCH
published: 28 July 2020
doi: 10.3389/fphar.2020.01125
Biological Properties and Absolute
Configuration of Flavanones From
Calceolaria thyrsiflora Graham
1
1
1
2,3
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Ernesto Valdes , Cesar Gonzalez , Katy Dıaz , Yesseny Vasquez-Martınez
,
Carolina Mascayano4 , Claudia Torrent , Francisco Cabezas , Sophia Mejias ,
4
4
3
*
3
3
5
*
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Margarita Montoya , Marcelo Cortez-San Martın , Marcelo A. Muñoz
,
Pedro Joseph-Nathan6, Mauricio Osorio1 and Lautaro Taborga
*
1
*
1
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Laboratorio de Productos Naturales, Departamento de Quımica, Universidad Tecnica Federico Santa Marıa, Valparaıso,
Edited by:
Marcello Locatelli,
2
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Chile, Programa Centro de Investigaciones Biomedicas Aplicadas, Facultad de Ciencias Medicas, Escuela de Medicina,
3
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Universidad de Santiago de Chile, Santiago, Chile, Departamento de Biologıa, Facultad de Quımica y Biologıa, Universidad
University of Studies G. d’Annunzio
Chieti and Pescara, Italy
4
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de Santiago de Chile, Santiago, Chile, Departamento de Ciencias del Ambiente, Facultad de Quımica y Biologıa,
Universidad de Santiago de Chile, Santiago, Chile, 5 Facultad de Ciencias, Instituto de Ciencias Qu´ımicas, Universidad
Reviewed by:
Sonia Cecilia Sgroppo,
6
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Austral de Chile, Valdivia, Chile, Departamento de Quımica, Centro de Investigacion y de Estudios Avanzados del Instituto
´
Politecnico Nacional, Mexico City, Mexico
National University of the Northeast,
Argentina
´
Marıa Daniela Santi,
Flavanones (–)-(2S)-5,4’-dihydroxy-7-methoxyflavanone (1) and (–)-(2S)-5,3’,4’-
trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria
thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of
Chile. The absolute configuration of these compounds was resolved by optical rotation
experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do
structure-activity relationships with the biological assays studied. Biological tests revealed
that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-
resistant strain S. aureus MRSA 97-77 (MIC value of 50 µg/ml). In addition, flavanone 2
showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the
traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract.
Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 1.61 µM) but 4.6-
and 17-fold lesser activity than etoposide and taxol.
´
Agencia Nacional de Investigacion e
´
Innovacion, Uruguay
*Correspondence:
Carolina Mascayano
carolina.mascayano@usach.cl
Marcelo A. Muñoz
mamunozflores@uach.cl
Mauricio Osorio
mauricio.osorio@usm.cl
Lautaro Taborga
lautaro.taborga@usm.cl
Specialty section:
This article was submitted to
Ethnopharmacology,
a section of the journal
Keywords: Calceolaria thyrsiflora, flavanone, absolute configuration, anti-inflammatory activity,
antibacterial activity
Frontiers in Pharmacology
Received: 22 April 2020
Accepted: 10 July 2020
Published: 28 July 2020
INTRODUCTION
Citation:
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Valdes E, Gonzalez C, Dıaz K,
The South-American geography allows the growth of a great flora diversity with a high degree of
endemism (Villagran and Hinojosa, 1997) where different Calceolaria species are found. This genus
of the spermatophytes belongs to the Angiosperms division and is found from Patagonia to central
Mexico, with most species growing near the Andes mountains (Molau, 1988). Thus, there are
around 50 species in Chile, of which some have been used in the traditional Chilean medicine due to
their antibacterial, diuretic, and digestive properties (Garbarino et al., 2007).
´
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Vasquez-Martınez Y, Mascayano C,
Torrent C, Cabezas F, Mejias S,
´
Montoya M, Cortez-San Martın M,
Muñoz MA, Joseph-Nathan P,
Osorio M and Taborga L (2020)
Biological Properties and Absolute
Configuration of Flavanones From
Calceolaria thyrsiflora Graham.
Front. Pharmacol. 11:1125.
One of them, C. thyrsiflora Graham, that grows in steep, sunny, and eroded places, mainly in
streams (Ministerio de Salud de Chile, 2010) blooms from September to December, is found
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between the provinces of Coquimbo and Curico, both on coast and inland locations, and is also
doi: 10.3389/fphar.2020.01125
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Flavanones From Calceolaria thyrsiflora
abundantly found in slopes of the surroundings of Santiago
(Ehrhart, 2000). It is commonly known as “Herb or Sweet Stick”
due to the intense dominant flavor of its leaves, whose infusion
has healing effects and is popularly used to relieve sore throats,
tonsils inflammation, and lesions of the oral mucosa (thrush and
gum inflammation). Due to its astringent and diuretic properties,
it is also used for the treatment of urinary incontinence and other
renal or bladder conditions, as well as a sweetener to flavor
drinks (Erazo Giuffra et al., 1987; Ministerio de Salud de Chile,
2010). Other studies on several Calceolaria species account for
antimicrobial, antioxidant (Falcão et al., 2006), and insecticidal
activities (Cespedes et al., 2016), while chemical studies allowed
the characterization of aromatic, diterpene, triterpene, glycoside,
obtained with hexanes/ethyl acetate 13:7 and 3:2 were separately
crystallized using hexanes/ethyl acetate with high temperature.
Chemical Assays
Estimation of the Total Flavonoid Content
The total flavonoid content was determined using the Dowd
method as described (Arvouet-Grand et al., 1994), with
modifications to adapt the screen of 96-well plates. Briefly, 150
µl of 2% AlCl3 in ethanol was mixed with the same volume of
extract solution in ethanol (1.0 mg/ml). Absorption readings at
415 nm were taken after 10 min against a blank sample consisting
of a 150 µl extract solution with 150 µl methanol without AlCl3.
The total flavonoid content was determined using a standard
curve with quercetin (0–70 mg/L) as the standard (Y = 71.545 x -
0.6373, R2 = 0.9994). The mean of three readings was used and
expressed as mg of quercetin equivalents (mg QE) per g of extract.
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and flavonoid secondary metabolites (Cespedes et al., 2013).
Herein we describe the antibacterial, antiproliferative, anti-
inflammatory (inhibition of 5-hLOX), and antifungal effects of
extracts of C. thyrsiflora and of the two isolated metabolites
(–)-(2S)-5,4’-dihydroxy-7-methoxyflavanone (1) and (–)-(2S)-
5,3’,4’-trihydroxy-7-methoxyflavanone (2), as well as the
spectroscopic characterization and absolute configuration
assessment of the two secondary metabolites by electronic and
vibrational circular dichroism. In addition, the three analogous
racemic flavanones 3, 4, and 5 were synthesized to evaluate their
biological activity.
Compound 1, also known as sakuranetin, was first isolated
from the Japanese cherry tree Puneus pseudo-cerrasus (Asahina,
1908) in the early 20th century, while compound 2 was first
identified as a constituent of South-American Tessaria
dodoniefolia in 1973 (Kavka et al., 1973). Most recently, 1 and
2 have been reported as constituents of Siolnatria brasilensis (dos
Santos et al., 2019), Rhus retinorrhoea (Mossa et al., 1996) and
Arteminia halodendron (Sun et al., 2019).
Estimation of the Total Phenolic Content
A modified Folin-Ciocalteu (FC) method (Madrid et al., 2015) was
used. The sample solution (15 mL) was mixed with distilled water
(15 mL) and 0.2 N FC reagent (150 mL). After 5 min, the 3% sodium
carbonate solution (120 mL) was added. A mixture of distilled water
(15 mL), methanolic sample solution (15 mL), HCl 0.2 N (150 mL),
and 3% sodium carbonate solution (120 mL) was used as blanks. The
absorbance was measured at 765 nm. The mean of three readings
was used and expressed as gallic acid equivalents (GAE; mg/g
extract) using a gallic acid (0–500 mg/L) standard curve.
Statistical Analysis
All data were expressed as the mean standard deviation (SD).
Graphpad Prism 7 software was used to ascertain statistically
significant differences. The differences among multiple groups were
evaluated using the one-way analysis of variance non-parametric
(Kruskal-Wallis test) with Dunn’s post-test. The difference between
the means was considered statistically significant at P < 0.05.
MATERIALS AND METHODS
Spectroscopic Analysis
Plant Material
IR spectra were obtained from KBr pellets on a Thermo Scientific
Nicolet 6700 FT-IR spectrophtometer (Waltham, MA, United
States), MS analysis were measured on a Thermo Fisher Scientific
The leaves and branches of C. thyrsiflora were collected during
the flowering stage from Cuesta Zapata, Melipilla province,
Santiago (33° 24’ 01.4’’ S, 71° 15’ 20.7’’ O) in December 2015.
The species was recognized by forestry engineer Patricio Novoa
Quezada of the National Forestry Corporation. A voucher
specimen (001016) is in deposit at the Herbarium of
™
Exactive Plus Orbitrap high-resolution mass spectrometer
(Bremen, Germany) at a resolution of 140,000 amu (10% valley),
and NMR spectra were measured on a Bruker AC-400 spectrometer
(Rheinstetten, Germany) at 400 MHz for 1H and 100 MHz for 13C in
CDCl3 for 1 and in DMSO-d6 for 2, Spectra were referenced using
the residual solvent peaks at d 7.26 (CDCl3) and d 2.50 (DMSO) for
1H and at d 77.0 and 39.5 for 13C, respectively.
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Universidad Tecnica Federico Santa Marıa, Valparaiso, Chile.
Extraction and Isolation of Flavanones
The aerial parts of C. thyrsiflora were dried at room temperature
under protection of sun light for two days. The 3.1 kg of dry and
ground plant material was successively extracted on an Edwards
and Ceruti stainless steel macerator with two liters of hexanes,
dichloromethane, and ethanol for 72 h. The solvents were
evaporated under reduced pressure to leave viscous residues.
A sample of 15 g of the dichloromethane extract was column
chromatographed on silica gel 60 eluted with hexanes/ethyl
acetate (1:1). HF-254 silica gel TLC plates were used to
monitor the separation, detecting spots under UV light and by
heating after drenching with 10% H2SO4 in H2O. The fractions
Stereochemical Analyses
HPLC-CD-OR Analysis
Electronic circular dichroism and optical rotation analyses were
made using a Shimadzu HPLC chromatograph coupled to a
JASCO CD-2095plus CD detector and a PDR advanced laser
polarimeter detector, and data were acquired using JASCO
ChromNAV software version 2.01.05. Chiroptical measurements
were obtained through a chromatographic analysis using a
Phenomenex Lux Cellulose-1 column (150 mm x 4.5 mm, 5 µm)
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with a mobile phase composed of n-hexane/isopropanol 9:1 at a flow
rate of 1.0 ml/min. Optical rotation response was measured at 670
nm, while UV and ECD spectra were measured between 220 and
420 nm by stop-flow while each compound was in the CD detector
cell. Although this HPLC-CD-OR measurement methodology was
not used for the quantitative analyses of the pure isolated flavanones,
a validation procedure was performed using pure racemic flavanone
and obtaining consistent results with those described in a previous
work (Muñoz et al., 2013).
up the mixtures were recrystallized from ethanol to give the chalcones
as pure solids. Their melting point and spectral data are given below.
General Procedure for the Synthesis of Flavanones
3, 4, and 5
They were synthesized as described with some modifications
(Ketabforoosh et al., 2014). To a stirred solution of chalcone (1
equiv.) in EtOH (5 ml/mmol) were added NaOAc (7 equiv.) and
H2O (vol. equiv. of EtOH). The reaction mixtures were heated
under reflux for 6 h. and then allowed cooling to room
temperature. After work-up the flavanone mixtures were
purified by column chromatography using hexanes-ethyl acetate
mixtures of increasing polarity (Ketabforoosh et al., 2014). The
OMOM groups were hydrolyzed with 1N HCl in methanol under
reflux for 15 min longer hydrolysis times lowered yield.
VCD Measurements
IR and VCD spectra were measured on a BioTools ChiralIR FT
spectrophotometer equipped with dual photoelastic modulation. A
sample of 4.3 mg of derivative of 1 with 4’-O-acetyl (1AC) in 150 mL
of 100% atom-D CDCl3 was placed in a BaF2 cell with a path length
of 100 mm, data were acquired at a resolution of 4 cm-1 during 6 h,
1
Biological Assays
Fungal Strains and Culture Conditions
and the samples stability was monitored by 300 MHz H NMR
spectra immediately prior to and after the VCD measurements.
A strain of Botrytis cinerea isolated from a naturally infected
grape (Vitis vinifera) was used in all experiments. Isolated
Phytophtora cinnamomi was provided by the Agricultural
DFT Calculations
A systematic conformational search for 1AC was performed using
molecular mechanics as implemented in the Spartan’04 software
package (Wavefunction-Inc., 2004). The two conformers obtained
when considering a 10 kcal/mol energy cutoff above the global
minimum energy value, were submitted to geometry optimization
with the Gaussian 03W software package (Frisch et al., 2003)
using the B3LYP and B3PW91 hybrid functionals and the
DGDZVP basis set. The IR and VCD frequencies obtained at
the same level of theory for both low energy conformations were
weighted according to a Boltzmann distribution and scaled using
an anharmonicity factor of 0.972, which was estimated using the
CompareVOA software (Debie et al., 2011), and plotted as
Lorentzian bands with half-widths of 6 cm-1.
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and Livestock Service of Valparaıso. Both strains were
maintained on a potato dextrose agar (PDA) medium (DIFCO,
Detroit, MI) at 4°C. The inoculum of both pathogens was grown
on PDA and incubates in dark at 23°C for 5 days in the case of B.
cinerea and 8 days for P. cinnamomi.
In Vitro Fungicidal Activity Assays Against B. cinerea
and P. cinnamomi
The fungicidal activity of flavanones, extracts, the negative control
(C-) composed of solvent (1% ethanol), and the positive control
(C+, commercial fungicide Captan® and metalaxil® for B. cinerea
and P. cinnamomi, respectively) were assessed using the radial
growth test technique on PDA medium as described (Taborga
et al., 2015). All test samples were dissolved in ethanol (1%) and
water, to obtain final concentrations of 50, 150, and 250 mg/L in
the PDA medium. A plug (4 mm) of PDA medium with five-day-
old mycelium colonies of the pathogen was placed at the center of
a Petri dish with PDA medium with or without studied test
samples and were incubated at 23°C in dark for 72 h (B. cinerea)
and 120 h (P. cinnamomi). After incubation, the diameters of
colonies were measured and the inhibition percentage was
calculated as the average of three replicas compared to C-.
Synthesis of Flavanone Analogues 3, 4,
and 5
Chemicals
All purchased chemical reagents (Merck or Sigma-Aldrich) were of
the highest available purity and used without previous purification.
Melting points (mp in °C) were measured on a Stuart-Scientific
SMP3 melting point apparatus and are uncorrected.
General Procedure for the Protection of Phenolic
Compounds With MOMCl
In Vitro Antibacterial Activity Assays,
Phytopathogens
The minimum inhibitory concentrations (MIC) were determined
All protected phenolic compound were synthesized as described
(Hyun et al., 2010) with some modifications. To a stirred solution
of phenolic compound (1.0 equiv.) in CH2Cl2 (0.12 M
concentration) were added i-Pr2NEt (1 equiv. per OH group)
and MOMCl (1 equiv. per OH group) dropwise at 0°C, and the
reaction mixture stirred at room temperature until the reaction
completed. The 1H, 13C-NMR and 2D NMR data were consistent
with the reported structures (Khupse and Erhardt, 2007; Du
́
using the broth microdilution method (Dıaz et al., 2018). Two-
fold serial dilutions were added to the wells of sterile 96-well plates
™
containing inoculated BBL Mueller Hinton II Broth medium
suspensions of bacterial cells (1.0 x105 UFC/ml) of Agrobacterium
tumefaciens and Bacillus subtilis. The final concentrations ranged
from 12.5 to 1,600 µg/ml. Ethanol (1%) was used as the negative
control and chloramphenicol (CALBIOCHEM; U.S. and Canada)
was used as the positive control against both bacteria. MICs were
determined according to the OD600 obtained in a Thermo
Scientific Multiskan GO 96-well plate photometer. After the
cultures were incubated at 37°C for 24 h, MIC was confirmed as
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et al., 2017; Barbuscakova et al., 2018).
General Procedure for the Synthesis of Chalcones
They were synthesized as described (Ndoile and Heerden, 2013) with
some modifications. The reaction mixture was warmed to 50°C and
stirred until completion of reaction as monitored by TLC. After work-
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the lowest concentration that completely inhibited the bacteria
growth. Assays were done in triplicate.
In Vitro 5-hLOX and 15-sLOX Enzyme Assays
The commercially available enzyme 5-hLOX from Cayman
Chemicals Inc. (Ann Arbor, MI, USA), was diluted (1:500) in the
assay buffer. This contained 4-(2-hydroxyethyl)-1-piperazineethane
sulfonic acid (HEPES) 50 mM, ethylenediamine tetraacetic acid
(EDTA) 2 mM, adenosine triphosphate (ATP) 10 mM, and CaCl2
10 mM at pH 7.5) admixed with 10 mM of H2DCFDA. The reaction
mixture was incubated for 15 min in the assay plate. Later, 280 mL of
buffer and 10 mM of inhibitors were added per well and incubated
for 30 min at 37°C. Finally, the reaction was started by adding a
suitable concentration of arachidonic acid (0.5 mM) and the
In Vitro Antibacterial Activity Assays, Human
Pathogens
Two clinical isolates of methicillin resistant S. Aureus (622-4 and
97-7), and one clinical isolate of E. coli 33.1, kindly donated by Dr.
Marcela Wilkens from Universidad de Santiago de Chile were used,
and S. aureus (NCTC8325-4) and E. coli (ATCC25922) were used
as the control strains. The antimicrobial activities of the isolated
compounds against E. coli (ATCC25922), E. coli multi-resistant
(33.1), S. aureus (NCTC8325-4), and methicillin-resistant S. aureus
97-7 strains, were determined using the microdilution method
established by CLSI (NCCLS, 1999). Briefly, stock solutions (5 mg/
ml) of compounds in DMSO were diluted in Mueller-Hinton broth
(MHB) to the different two-fold assay concentrations. The final
concentration of DMSO was ≤ 2.5% and does not affect the
microbial growth. The obtained solution was added to MHB and
serially two-fold diluted in a 96-well microplate. A sample of 100
mL of inoculum 1.5 × 106 colony forming unit (CFU) ml-1 in MHB
was added and then incubated at 37°C for 18 h. The assay was
repeated three times. Wells containing MHB, 100 mL of inoculum,
and DMSO served as negative controls. The MIC was defined as
the lowest concentration of compounds resulting in the complete
inhibition of visible growth (Felix et al., 2009).
™
fluorescence was read in a multimode detector Synergy HT
Multi-Mode Microplate Reader (Biotek, Bad Friedrichshall,
Germany) at 480 nm exciting at 520 nm after incubation at room
temperature for 1 h. The IC50 values were obtained for analysis of
oxidation of H2DCFDA dye to the highly fluorescent 2’,7’-dichloro-
fluorescein (DCF) using the non-linear curve-fitting program. The
kinetic assays to determine the mechanism of inhibition were done
with the best inhibitors, using different concentrations of the
inhibitor (0–1 mM) and substrate (0.2–5 mM). All data were
collected in duplicate and the assays were performed on different
days to ensure reproducibility.
The activity of 15-sLOX (Cayman Chemical Item No. 60712)
was determined following the formation of reaction products at
234 nm (ϵ = 25,000 M-1 cm-1) in a Perkin-Elmer Lambda 25 UV/
Vis spectrophotometer. All reactions were performed at a final
volume of 2 ml and were constantly stirred using a magnetic bar at
room temperature. The reaction medium used contained 25 mM
HEPES buffer (pH 7.4), Triton X-100 0.01%, and the arachidonic
acid (AA) substrate at a 10 µM concentration determine
preliminary percent inhibition (%I). AA concentration was
determined quantitatively by allowing the enzymatic reaction to
go to completion. The reaction was carried out by adding the
inhibitor dissolved in DMSO (5 µg/ml in the reaction medium) to
the bucket with the substrate buffer, and finally the enzyme was
added. Assays were performed in duplicate on three different days.
GraphPad Prism Demo V.7 performed the graphics for IC50 and
kinetics studies (GraphPad-Software-Inc., 2017).
In Vitro Cytotoxic Activity of Flavanone 2
The used cell lines include MDA-MB-231 human breast
adenocarcinoma cells, B16-F10 mouse metastatic melanoma cells
and MEF primary mouse embryonic fibroblast. Cells were
maintained in a DMEM high glucose medium (Mediatech,
Manassas, VA, USA) supplemented with 10% (MDA-MB-231 and
B16-F10) or 15% (MEF) heat-inactivated fetal bovine serum
(HyClone Laboratories, South Logan, USA), 100 IU ml-1 penicillin
and 100 mg ml-1 streptomycin while maintained at 37°C in a 5% CO2
humidified atmosphere. Cell viability was measured using a
CyQuant® Direct Cell Proliferation Assay Kit (Life Technologies,
Grand Island, NY, USA) following the manufacturer’s instruction.
Briefly, 5,000 cells per well were seeded onto a flat-bottom 96-well
plate in 200 mL final volume. Six hours after seeding, the culture
medium was replaced by the medium containing the tested
compounds at 0.01 to 100 µM concentrations in DMSO (0.1%
final concentration) during 72 h. Untreated cells (medium
containing only 0.1% DMSO) were used as controls. At the end of
the incubation, 100 mL of culture medium was removed from each
well and replaced by detection reagent solution provided by the kit
(which is a 1:5 mixture of the reagent CyQUANT®, direct nucleic
acid stain and the reagent labeled as CyQUANT® direct background
suppressor I). Cells were incubated for 1 h and fluorescence emission
was measured at 535 nm with excitation at 480 nm in a microplate
reader (Infinite 200 PRO, Tecan, Grödig, Austria). At least four
independent experiments were performed for each concentration.
The results from each experiment were transformed into percentage
of controls and the LC50 values were graphically obtained from the
dose-response curves. The LC50 value was obtained adjusting
the dose-response curve to sigmoidal curves (variable slope) using
the GraphPad Prisma 6.0 software (GraphPad-Software, 2015).
Docking of Flavanone 2 With 5-hLOX
The structure of 2 was built with the Molecular Operating
Environment software (Chemical-Computing-Group-ULC,
2018). ChelpG charges were obtained at the B3LYP/6-31G(d,p)
level of theory, employing the Gaussian 09 software (Frisch et al.,
2016). Docking of all inhibitors into the active site of the crystal
structure of 5-hLOX (PDB code: 3O8Y, 2.39 Å resolution) was
performed with the AutoDock4 package (Morris et al., 2009)
using a Lamarckian algorithm and assuming total flexibility of
the inhibitors, the docking validation was done with known
inhibitors of 5-hLOX because doesn’t exist co-crystallized
inhibitors with the enzyme. The grid maps were made up to
60 × 60 × 60 points, with a grid-point spacing of 0.375 Å and the
Fe3+ as the center of the grid map. The AutoTors option was
used to define the ligand torsions, and the docking results were
then analyzed by a ranked cluster analysis, resulting in
conformations with the highest overall binding energy (most
negative Gibbs free energy binding value, –DG).
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1H NMR (DMSO, 400 MHz) d: 12.1 (s, 1H, OH-5); 9.14 (s, 2H,
OH-3’, OH-4’); 6.92 (s, 1H, H-2’); 6.78 (s, 2H, H-5’, H-6’); 6.11
(dd, 2H, J = 2.0, 6.7 Hz, H-6, H-8); 5.45 (dd, 1H, J = 2.8, 13 Hz,
H-2); 3.82 (s, 3H, OCH3); 3.27 (dd, 1H, J = 13.0, 17.0 Hz, H-3b);
2.75 (dd, 1H, J = 2.8, 17.0 Hz, H-3a). 13C NMR (DMSO, 100
MHz) d: 197.0 (C-4); 167.5 (C-7); 163.3 (C-5); 162.9 (C-9); 145.8
(C-3’); 145.3 (C-4’); 129.4 (C-1’); 118.1 (CH-6’); 115.4 (CH-5’);
114.4 (CH-2’); 102.7 (C-10); 94.7 (CH-6); 93.9 (CH-8); 78.7
(CH-2); 56.0 (OCH3); 42.2 (CH2-3). MS m/z: 299.0566,
301.0721, 302.0753. The spectra data were consistent with the
reported structure (Mossa et al., 1996).
RESULTS AND DISCUSSION
Chemical Activity
Extracts, Total Flavonoids, Total Phenolics Content,
and Compounds Isolated From C. thyrsiflora
The hexanes, dichloromethane, and ethanol extracts yielded 0.57, 2.5,
and 5.2% residues, respectively. Total flavonoids and total phenolic
content compounds were measured. The results are shown in
Table 1. The polar extracts ED and EE had significantly higher
total flavonoid and total phenolic compound values than the less
polar EH extract. ED extract had a total flavonoid value higher than
EE (62.02
0.15 and 46
1.53 mg eq. quercetin/g extract,
Absolute Configuration of 1 and 2 Through
Chiroptical Spectroscopy and DFT
respectively). For this reason, ED was subjected to column
chromatography separation and crystallization of the fractions,
flavanones 1 and 2, as white crystals, were obtained in 4.59 and
1.08% yields, respectively. These flavonoid aglycones 1 and 2 were
isolated from ED extract, which is expected for this solvent
(Grotewold, 2006).
The absolute configuration determination of 1 and 2 was
ignored in a previously study (Ley et al., 2005) since no optical
rotation was accounted.
Previous characterization of the secondary metabolites of C.
thyrsiflora focused on the hexanes, dichloromethane, and ethyl
acetate extracts demonstrating the presence of diterpenes
(Chamy et al., 1991) and glycosides (Di Fabio et al., 1995).
Aromatic compounds and alkaloids have also been extracted
from this species and evaluated with their respective
antimicrobial activity (Bravo et al., 2005).
Calculations
Compounds 1 and 2 share the common C-2 stereogenic center
and can exist as two enantiomers. As a first step to assign the
absolute configuration (AC) of both compounds, optical rotation
(OR) and electronic circular dichroism (ECD) measurements
were performed using suitable chromatographic detectors
coupled to a high-performance liquid chromatograph (HPLC)
in a serial arrangement (HPLC-ECD-OR). These measurements
showed that both compounds are levorotatory and exhibit nearly
identical ECD spectra in the 220–420 nm spectra region (Figure 1).
These observations clearly suggest that both flavanones possess the
same AC as they differ only in the presence of an additional
hydroxy group at C-3´ in 2. A literature search reveals that (-)-1
has been associated to the (S) enantiomer (Jerz et al., 2005), which
suggests both compounds have this AC. The above assumption is
further corroborated when ECD traces of both natural products are
compared with that of parent (-)-(S)-flavanone for which also a
very similar spectrum is observed (Figure 1).
Although ECD is widely accepted to establish the absolute
configuration of the sole stereogenic center of flavanones, the
current study provided the first opportunity to explore vibrational
circular dichroism (VCD) spectroscopy for such a task using natural
occurring flavanones of known absolute configuration. ECD generally
provides a very limited number of informative transitions in the UV
region, while VCD provides many bands in the IR region, and
therefore VCD is a significantly more robust methodology than ECD.
In addition, VCD is not dependent on the use of model compounds
as is implicit in ECD studies of flavanones. VCD has shown to be a
reliable technique for the assignment of the AC of secondary
metabolites (Burgueño-Tapia and Joseph-Nathan, 2015; Joseph-
Spectroscopic Characterization of Isolated
Compounds
The isolated compounds correspond were identified as (-)-(2S)-
5,4’-dihydroxy-7-methoxyflavanone (1) and (-)-(2S)-5,3’,4’-
trihydroxy-7-methoxyflavanone (2).
(-)-(2S)-5,4’-Dihydroxy-7-methoxyflavanone (1). HRMS m/z,
observed: 285.0769; C16H14O5 [M - H]- requires: 285.0768. IR
(KBr) Vmax cm-1: 3529, 3457, 3147, 1630, 1618, 1570. H NMR
1
(CDCl3, 400 MHz) d: 12.0 (s, 1H, OH-5); 7.34 (d, 2H, J = 8.5 Hz, H-2’,
H-6’); 6.89 (d, 2H, J = 8.6 Hz, H-3’, H-5’); 6.07 (d, 1H, J = 2.3 Hz, H-
6); 6.04 (d, 1H, J = 2.3 Hz, H-8); 5.36 (dd, 1H, J = 3.0, 13.0 Hz, H-2);
3.81 (s, 3H, OCH3); 3.09 (dd, 1H, J = 13.0, 17.0 Hz, H-3b); 2.78 (dd,
1H, J = 3.0, 17.0 Hz, H-3a). 13C NMR (CDCl3, 100 MHz) d: 196.0 (C-
4); 168.0 (C-7); 164.1 (C-5); 162.9 (C-9); 156.1 (C-4’); 130.5 (C-1’);
128.0 (CH-2’, 6’); 115.7 (CH-3’, 5’); 103.1 (C-10); 95.1 (CH-6); 94.2
(CH-8); 79.0 (CH-2); 55.7 (OCH3); 43.2 (CH2-3). The spectra data
were consistent with the reported structure (Vasconcelos et al., 1998).
(-)-(2S)-5,3’,4’-Trihydroxy-7-methoxyflavanone (2). HRMS
m/z, observed: 301.0721; C16H14O6 [M - H]- requires:
301.0718. IR (KBr) Vmax cm-1: 3370, 3183, 1638, 1603, 1572.
́
Nathan and Gordillo-Roman, 2015) including flavanones (Muñoz
et al., 2013). Nevertheless, such studies typically require a 5–10 mg
sample to be dissolved in 150 µl of CDCl3 or CCl4. In the cases of 1
and 2, such requirements are not meet due to the poor solubility of
either sample in these solvents, and therefore derivatives of polar OH
TABLE 1 | Total flavonoids and phenolics content of C. thyrsiflora extracts.
Extract EH
Extract ED
Extract EE
Total flavonoids (mg eq. quercetin/g extract SD)
Total phenolic content compounds (mg eq. gallic acid/g extract SD)
11.51 0.21a
18.28 1.68
62.02 0.15b
141.73 3.04
46.77 1.53c
150.40 11.92
ap< 0,05 EH versus ED; bp< 0,05 ED versus EE; cp< 0,05 EH versus EE (Kruskal-Wallis test, Dunns post-test for multiple comparison).
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Flavanones From Calceolaria thyrsiflora
FIGURE 1 | Comparison of the ECD spectra of (-)-(S)-1 (dashed line), (-)-(S)-
2 (dotted line), and (-)-(S)-flavanone (continuous line).
groups to generate acetyl or methoxy containing molecules can
overcome such inconveniences thereby allowing successful
measurements. In the present case, only 1 was available in the
required amounts and thus its treatment with acetic anhydride
provided the 4’-O-acetyl derivative 1AC (Figure 2). Accordingly,
the VCD and IR spectra of the later were measured and compared in
silico with calculated spectra for the (S) enantiomer (Figure 3). These
calculations required a conformational search using the MMX
molecular mechanics force field, which afforded only two low
energy conformations differing in the C-6–C-7–O–CH3 dihedral
angle. Both conformers were subjected to geometry optimizations
and vibrational calculations using density functional theory (DFT) at
the B3LYP/DGDZVP level of theory. The comparisons shows with a
100% confidence level (Debie et al., 2011), that 1AC has the (S) AC.
Visual comparison of the VCD spectrum of 1AC with those
of (R)- and (S)-flavanone reported by us (Muñoz et al., 2013),
unexpectedly showed a better agreement with the former than
with the later enantiomer. This odd observation induced us to
revise the VCD spectra of the enantiomers of parent flavanone,
which revealed the published spectra (Muñoz et al., 2013) are
wrongly labeled. The correct spectra of the two flavanone
enantiomers are those presented herein, which when
compared with that of 1AC (Figure 3), clearly shows
agreement with the (S) enantiomer, as expected, and therefore
confirms that 1 and 2 share the (S) AC as deduced using the OR
and ECD observations.
FIGURE 3 | Comparison of the observed VCD spectrum of (-)-(S)-1AC with
the B3LYP/DGDZVP calculated spectrum for the (S) enantiomer, and the
observed spectra of (S)- and (R)-flavanone. The calculated spectra include an
anharmonicity factor of 0.972.
Synthesis of Flavanones Analogs 3, 4,
and 5
The synthesis of analogs of the isolated compounds was done to
determine the influence of the hydroxy and methoxy groups
positions on the biological activity. Thus, Claisen-Schmidt
condensation, cyclization under mild alkaline conditions, and
acidic hydrolysis as shown in Figure 4 was undertaken. The
reaction of the acetophenone with different benzaldehydes (3B,
4B, and 5B) in alkaline media produced chalcones (3C, 4C, and
5C) in excellent yields (86–98%). Further cyclization and
hydrolysis reactions formed flavanones in regular to good
yields (23–69%). However, longest hydrolysis times gave
poorer yields.
Spectroscopic Characterization of
Chalcones
The synthesized chalcones are 3C, 4C, and 5C.
3-(3,4-bis(Methoxymethoxy)phenyl)-1-(2-hydroxy-4,6-bis
(methoxymethoxy)phenyl)prop-2-en-1-one (3C) was obtained
as a yellow solid, m.p. 93 - 94°C as reported (Detsi et al., 2009);
FIGURE 2 | Compounds studied by ECD.
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FIGURE 4 | Preparation of flavanones (3, 4, and 5) via Claisen-Schmidt condensation in alkaline medium of acetophenone A and benzaldehydes B (i: KOH/MeOH/
H2O) to produce chalcones C, cyclization of chalcones C under mild alkaline conditions (ii: NaOAc, EtOH-H2O, reflux), and acid hydrolysis (iii: HCl 1N, MeOH, reflux)
to obtain flavanones F. Reaction yields are shown in brackets.
1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)-3-(3-methoxy-
4-(methoxymethoxy)phenyl)prop-2-en-1-one (4C) was obtained
as a yellow solid, m.p. 83 - 84°C as reported (Duan et al., 2006);
and 1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one (5C) was obtained as a yellow
solid, m.p. 97–98°C as reported (Alam and Islam, 2003).
Biological Assays
In Vitro Fungicidal Activity of Extracts and Isolated
Compounds
Table 2 shows that all concentrations of the dichloromethane
extract (ED) have high activity only against P. cinnamomi, while
only hexanes extracts (EH) exhibit inhibitory activity against B.
cinerea. None of these extracts showed significant activity
compare to Captan and Metalaxil. In turn, only the ED extract
showed activity against P. cinnamomi; although neither 1 nor 2
explain the activity of the extract. Due to the low activity of the
extract, the compounds were not analyzed against B. cinerea.
There are no reported studies of antifungal activity of extracts or
compounds isolated from C. thyrsiflora.
Spectroscopic Characterization of
Flavanones
The synthesized flavanones are 3, 4, and 5.
5,7,3’,4’-Tetrahydroxyflavanone [( )-eriodictyol] (3) was obtained
as white solid (0.86 g, 69%); mp: 265,3 – 267,1°C as reported (Wurm
and Geres, 1982); 3’-methoxy-4’,5,7-trihydroxyflavanone
[( )-homoeriodictyol] (4) was obtained as a white solid (0.64 g,
46%); mp: 225,6 - 227,3°C as reported (Zhao et al., 2011); 5,7-
dihydroxyl-4’-methoxyflavanone [( )-isosakuranetin] (5) was
obtained as a white solid (0.35 g, 23%); mp: 195–195.5°C as
reported (Horowitz and Gentili, 1961) for (-)-isosakuranetin, and
(Zhao et al., 2011) for [( )-isosakuranetin]. All NMR spectra
were consistent with those reported.
In Vitro Antibacterial Effects of Pathogenic Bacteria
to Plant Extracts and Metabolites
EH and ED showed high activity against both phytopathogenic
bacteria, unlike the more polar extract EE (Table 3) but lower than
chloramphenicol but the isolated compounds have no antimicrobial
activity against the studied phytopathogenic bacteria.
TABLE 2 | Antifungal activity of extracts and compounds isolated from C. thyrsiflora on in vitro mycelial growth of B. cinerea and P. cinnamomi.
Extract or compound
Percentage of inhibition on in vitro mycelial growth of B.
cinerea (%)
Percentage of inhibition on in vitro mycelial growth of P.
cinnamomi (%)
50 mg/L
150 mg/L
250 mg/L
50 mg/L
150 mg/L
250 mg/L
EH
ED
EE
1
0.0 0.0
0.0 0.0
0.0 0.0
N.D
N.D
94 5.2
–
62.0 2.9
19.0 1.0
0.0 0.0
N.D
N.D
100 0.1
–
70.0 5.6
36.0 0.1
0.0 0.0
N.D
N.D
100 0.2
–
0.0 0.0
64.0 0.6
0.0 0.0
0.0 0.0
0.0 0.0
–
0.0 0.0
86.0 0.9
0.0 0.0
0.0 0.0
33.0 5.8
–
0.0 0.0
91.0 0.7
30.0 9.8
0.0 0.0
42.0 5.3
–
2
Captan
Metalaxil
100 0.2
100 0.1
100 0.1
N.D, Not determined.
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Flavanones From Calceolaria thyrsiflora
TABLE 3 | Antibacterial activity of extracts and compounds isolated from C.
thyrsiflora against pathogenic bacterial strains.
TABLE 5 | Cytotoxic activity of constituents of C. thyrsiflora.
Compound
LC50 (µM) SD
B16-F10
Extract or compound
Minimum inhibitory concentration (MIC) µg/ml
MDA-MB-231
MEF
GN A. tumefaciens
GP B. subtilis
1
2
∼ 1100
72.02 7.16
8.07 1.61
0.48 0.08
1.74 0.32
145.6 23.4
31.01 3.69
0.40 0.07
1.03 0.17
EH
ED
EE
1
50
50
200
N.A
N.A
12.5
50
50
400
N.A
N.A
12.5
48.72 9.32
0.21 0.48
1.33 0.28
Taxol
Etoposide
2
to the effect on non-cancerous cells (MEF). Flavanone 2 also
exhibited cytotoxic activity against MDA-MB-231, but with no
selectivity over MEF. When the cytotoxic effect of 2 against B16-
F10 was compared with commonly used chemotherapeutic drugs,
it showed 4.6- and 17-fold less activity than etoposide and taxol,
respectively. These activity have shown to be important for cancer
cells proliferation and viability (Ghosh and Myers, 1998;
Steinhilber et al., 2010; Chen et al., 2016) and we previously
showed that B16-F10 are more sensitive to drugs that interfere
Chloramphenicol
G.N, Gram-Negative Bacteria; G.P, Gram-Positive Bacteria; N.A, Not activity.
In Vitro Antibacterial Effects of Pathogenic Bacteria
to Humans
Testing the antibacterial activity of all isolated and synthesized
compounds against the resistant strains S. aureus NTCC 8325-4,
S. aureus MRSA 97-7, S. aureus MRSA 622-4, E. coli
ATCC25922, and E. coli 33.1 (Table 4) revealed that only 2
exhibited moderate inhibitory activity against methicillin-
resistant strain S. aureus MRSA 97-7, with a MIC value of 50
[µg/ml]. It is striking that this molecule has certain selectivity
towards the multiresistant strain MRSA 97-7, showing a lower
action towards the sensitive strain (NTCC8325-4), or towards
the other resistant strain MRSA 622-4. None of the molecules
showed activity against the Gram-negative strains (E. coli).
Previous studies evaluated the antibacterial activity of some
secondary metabolites extracted from C. thyrsiflora (Bravo et al.,
2005). The tested compounds were 2,4-dihydroxy-2H-1,4-
benzoxazin-3(4H)-one (DIBOA), 2-benzoxazolinone (BOA), 2-
dydroxy-2H-1,4-benzoxazin-3(4H)-one (HBOA), 2,7-
dihydroxy-2H-1,4-benzoxazin-3(4H)-one (7-OH-HBOA), and
gallic acid, showing inhibitory activity of bacterial growth in
high concentrations (1,000 µg/ml). The activity of these
compounds disappears at lower concentrations (250 µg/ml),
with the exception of gallic acid, which is the only molecule
possessing catecholic hydroxyls, achieving 67.2% inhibition
activity of S. aureus ATCC.
́
́
with 5-hLOX activity (Vasquez-Martınez et al., 2019).
In Vitro 5-hLOX and 15-sLOX Enzyme Assays
The enzymatic results (Table 6) showed that both flavones were
selective inhibitors of 5-hLOX. This difference could be
explained comparing the binding sites of LOX since is known
that the cavity of 5-hLOX is 20% larger than 15-sLOX (Mitra
et al., 2015) being dependent of molecular volume and shape and
the possibility to enter into the binding site. The IC50 results
showed that 2 was a more potent inhibitor (0.10 µM) than 1
(11.31 µM) for 5-hLOX and a structure evaluation confirms that
the catechol group is a key factor for the interaction with the
enzyme. Flavanone 2 showed the greatest 5-hLOX inhibitor
activity which correlated with a higher cytotoxic activity.
The above is confirmed with the kinetic results (Figures 5A,
B), showing that 2 is a competitive inhibitor (0.05 µM) of 5-
hLOX, meanwhile 1 presented a non-competitive mechanism
(4.71 µM). The potent in vivo anti-inflammatory effects of 2
found in several assays related to its neuroprotective action
(Fischer et al., 2019), could be in part explained by the
inhibition of 5-hLOX found in this work.
In Vitro Cytotoxic Activity of Flavanones 1 and 2
As in the previous case, Table 5 shows that 1 showed no
significant cytotoxic activity against MDA-MB-231 (human
breast adenocarcinoma cell line), B16-F10 (mouse melanoma
cells), or MEF (primary mouse embryonic fibroblast), while 2
exhibits a 4-fold higher selectivity towards B16-F10 as compared
Docking of Flavanone 2 With 5-hLOX
Docking studies were done for the best 5-hLOX inhibitor. The
results (Figure 6) showed that (S)-2 located inside the binding site
very near to the catalytic iron (energy binding -1.53 kcal/mol) in
accordance to a competitive mechanism, instead, we observed
TABLE 4 | Antibacterial activity of isolated and synthesized flavanones against resistant strains bacteria.
Compound
Minimum inhibitory concentration (MIC) µg/ml
S. aureusNTCC8325-4
S. aureusMRSA 97-7
S. aureusMRSA 622-4
E.coli ATCC25922
E.coli33.1
1
2
3
4
>100
>100
>100
>100
>100
3,5
>100
50
>100
>100
>100
25
>100
>100
>100
>100
>100
5
>100
>100
>100
>100
>100
–
>100
>100
>100
>100
>100
–
5
Vancomicin
Ampicillin
–
–
–
60
>100
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TABLE 6 | Biological activity of isolated and synthesized flavanones as 5-hLOX and 15-sLOX inhibitors.
Compound
% of inhibition(5-hLOX)
% of inhibition(15-sLOX)
3.125
IC50(5-hLOX)
Ki(5-hLOX)
Type of inhibition
1
2
3
4
61 1.341
93 0.791
88 0.678
11.67 2.45
2.57 2.26
93 5.870
7
11.31 µM 3.022
0.10 µM 0.081
0.22µM 0.023
N.D
4.71 µM 1.542
0.05 µM 0.012
0.07µM 0.013
N.D
non-competitive
competitive
competitive
N.D
N.D
competitive
26 3.955
N.D
N.D
N.D
N.D
5
N.D
N.D
N.D
NDGA
0.33 µM 0.15
Flavanones at 10 µM; IC50, Half inhibitory concentration; Ki, inhibitory constant; N.D, not determined; NDGA, nordihydroguaiaretic acid.
A
B
FIGURE 5 | (A) Michaelis-Menten for flavanone 2; (B) The Lineweaver-Burk graph of the inhibition of 5-hLOX by flavanones 1 and 2.
with ASN425 favoring the binding between 2 and 5-hLOX, also,
the structure shows hydrophobic interactions between the
aromatics rings of flavanone 2 and the residues as PHE, ILE,
and LEU, finally the cation-pi interaction is observed between the
flavanone and iron in the catalytic site, the same behavior is
observed for the other good inhibitor founded for 5-hLOX that is
flavanone 3 that confirm the relevance of the catechol group and
the inhibition of the enzyme.
CONCLUSIONS
In vitro fungicidal activity and antibacterial effects on bacteria
pathogenic to plants show that, although the ED has the best
activity, it is much lower than the controls used and the isolated
compounds are not responsible for this activity. Flavanone 2 has
selectivity activity in cytotoxic tests against B16-F10 and in the in
vitro antibacterial effects on bacteria pathogenic to human against
S. aureus MRSA 97-7. In the in vitro 5-hLOX enzyme assays,
flavanone 2 again has more activity than 1, probably due to the
presence of catecholic hydroxyls that is an important characteristic
founded in 5-hLOX inhibitors. This result validates the use in the
traditional medicine of the aerial parts of C. thyrsiflora as an anti-
inflammatory. The Docking study shows that the activity is related
to the presence of the 3’-OH group. However, when comparing 2
with 2, that although both compounds possess catecholic
hydroxyls, 2 has a higher activity probably due to its
stereochemistry. Finally, it should be noted that the (S) absolute
configuration of 1 and 2 was determined with certainty by ECD
and VCD for the first time. We have also verified that flavanone 1
is (S)-sakuranetin and 2 is (S)-sterubin. The docking study also
confirms this stereochemistry.
FIGURE 6 | Docking result of flavanone 2 and 5-hLOX.
positive binding energy with the flavanone 1 into the binding site
of the enzyme that agrees with the non-competitive mechanism.
We also studied the docking of the (R)-isomer whose results
showed not a good energy binding value (-0.42 kcal/mol). It was
noted that the 3’-OH of flavanone 2 forms a hydrogen bounding
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Flavanones From Calceolaria thyrsiflora
cytotoxic activity assays. All authors contributed to the article
and approved the submitted version.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher.
FUNDING
This work was supported by FONDECYT (No. 1130924, No.
́
1141071), PIIC-DGIIP of Universidad Tecnica Federico Santa
AUTHOR CONTRIBUTIONS
́
Marıa, DICYT-USACH (No. 021901VM, No. 021941MC) of
Universidad de Santiago de Chile, and CONACYT-Mexico
(No. 284194).
LT and MO designed the investigation. EV carried out the
extraction, separation, and purification of compounds. LT and
EV recollected the plant material and collaborated on the structure
determination of 1 and 2. CG collaborated on the structure
determination of 1 and 2. MO carried out the synthesis of 3–5
and evaluated the total flavonoids and total phenolic content. KD
performed the in vitro anti-phytopathogenic activity assays. MM
carried out the OR, ECD, and VCD calculations. PJ-N carried out
the VCD measurements and comparisons. YV-M and MC-M
performed the in vitro antibacterial activity assays against human
pathogenic bacteria and statistical analysis of the chemical activity.
CM and CT performed the in vitro 5-hLOX and 15-sLOX enzyme
assays and FC carried out the molecular docking studies with 5-
hLOX enzyme and 2. MM and SM carried out the in vitro
ACKNOWLEDGMENTS
́
The authors acknowledge Mr. Patricio Novoa from Corporacion
Nacional Forestal (CONAF), Viña del Mar, Chile.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fphar.2020.
01125/full#supplementary-material
Debie, E., Gussem, E., Dukor, R. K., Herrebout, W., Nafie, L. A., and Bultinck, P.
(2011). A Confidence Level Algorithm for the Determination of Absolute
Configuration Using Vibrational Circular Dichroism or Raman Optical
Activity. ChemPhysChem 12, 1542–1549. doi: 10.1002/cphc.201100050
Detsi, A., Majdalani, M., Kontogiorgis, C. A., Hadjipavlou-Litina, D., and Kefalas,
P. (2009). Natural and synthetic 2’-hydroxy-chalcones and aurones: Synthesis,
characterization and evaluation of the antioxidant and soybean lipoxygena.
Bioorg. Med. Chem. 17, 8073–8085. doi: 10.1016/j.bmc.2009.10.002
REFERENCES
Alam, S., and Islam, A. (2003). Synthesis of 2’ 4’-Dihydroxy-6’-Methoxy-3, 4-
Methylenedioxydihydro Chalcone and 2’, 4’, 6’-Trihydroxy-4-
Methoxydihydrochalcone. Pak. J. Sci. Ind. Res. 46, 27–30.
Arvouet-Grand, A., Vennat, B., Pourrat, A., and Legret, P. (1994). Standardisation
d’un extrait de propolis et identification des principaux constituants. J. Pharm.
Belg. 49, 462.468.
Asahina, Y. (1908). On Sakuranin, a New Glucoside in the Bark of the Japanese
Cherry Tree (Prunus pseudo-cerrasus, var. Sieboldi). Yakugaku Zasshi 313,
213–233.
Di Fabio, A., Bruni, A., Poli, F., Garbarino, J. A., Chamy, M. C., Piovano, M., et al.
(1995). The distribution of phenylpropanoid glycosides in Chilean calceolaria
Spp. Biochem. Syst. Ecol. 23, 179–182. doi: 10.1016/0305-1978(94)00088-X
̌
̌
́
́
́
́
́
̌
́
̌
́
̌
́
Barbuscakova, Z., Kozubıkova, H., Zalesak, F., Dolezal, K., and Pospısil, J. (2018). General
approach to neolignan-core of the boehmenan natural product family. Monatshefte
für Chemie - Chem. Mon. 149, 737–748. doi: 10.1007/s00706-017-2132-4
Dıaz, K., Espinoza, L., Madrid, A., Pizarro, L., and Chamy, R. (2018). Isolation and
Identification of Compounds from Bioactive Extracts of Taraxacum officinale
Weber ex F. H. Wigg. (Dandelion) as a Potential Source of Antibacterial Agents.
Evidence-Based Complement. Altern. Med. 8, 1–8. doi: 10.1155/2018/2706417
dos Santos, C. H. C., Talpo, T. C., Motta, B. P., Kaga, A. K., Baviera, A. M., Castro, R. N.,
et al. (2019). New compounds of Siolmatra brasiliensis and inhibition of in vitro
protein glycation damage. Fitoterapia 133, 109–119. doi: 10.1016/j.fitote.2018.12.023
Du, G., Zhao, Y., Feng, L., Yang, Z., Shi, J., Huang, C., et al. (2017). Design,
Synthesis, and Structure–Activity Relationships of Bavachinin Analogues as
Peroxisome Proliferator-Activated Receptor g Agonists. ChemMedChem 12,
183–193. doi: 10.1002/cmdc.201600554
́
Bravo, H., Copaja, S., Figueroa-Duarte, S., Lamborot, M., and San Martın, J.
(2005). 1,4-Benzoxazin-3-one, 2-Benzoxazolinone and Gallic Acid from
Calceolaria thyrsiflora Graham and their Antibacterial Activity. Z. für
Naturforsch. 60, 389–393. doi: 10.1515/znc-2005-5-604
Burgueño-Tapia, E., and Joseph-Nathan, P. (2015). Vibrational Circular
Dichroism: Recent Advances for the Assignment of the Absolute
Configuration of Natural Products. Nat. Prod. Commun. 10, 1785–1795. doi:
10.1177/1934578X1701200501
́
́
Cespedes, C. L., Salazar, J. R., and Alarcon, J. (2013). Chemistry and biological
activities of Calceolaria spp. (Calceolariaceae: Scrophulariaceae). Phytochem.
Rev. 12, 733–749. doi: 10.1007/s11101-013-9276-6
Duan, Y., Qi, Y., Ji, Z., Fang, G., Cheng, Y., and Wu, S. (2006). Synthesis of 2,3-
dihydro-5,7-dihydroxy-2-aryl-4H-1-benzopyran-4-one derivatives and
determination of their antibacterial and antimicrobial activity. (Zhongguo
Yaowu: Huaxue Zazhi Bianjibu) 16, 342–346.
Ehrhart, C. (2000). Die Gattung Calceolaria (Scrophulariaceae) in Chile.
(Bibliotheca Botanica).
Erazo Giuffra, S., Garcia Madrid, R., and Latorre De La Cruz, I. (1987). Native
plants used in Chilean folk medicine. Anales de la Real Academia de Farmacia.
53 (2), 296–301.
Cespedes, C. L., Lina-Garcia, L., Kubo, I., Salazar, J. R., Ariza-Castolo, A.,
Alarcon, J., et al. (2016). Calceolaria integrifolia s.l. complex, reduces feeding
and growth of Acanthoscelides obtectus, and Epilachna varivestis. A new
source of bioactive compounds against dry bean pests. Ind. Crops Prod. 89,
257–267. doi: 10.1016/j.indcrop.2016.05.026
́
Chamy, M. C., Piovano, M., Garbarino, J., Miranda, C., Gambaro, V., Rodrıguez,
M. L., et al. (1991). Diterpenoids from Calceolaria thyrsiflora. Phytochemistry
30, 589–592. doi: 10.1016/0031-9422(91)83731-Y
Falcão, D. Q., Costa, E. R., Alviano, D. S., Alviano, C. S., Kuster, R. M., and
Menezes, F. S. (2006). Atividade antioxidante e antimicrobiana de Calceolaria
chelidonioides Humb. Bonpl. & Kunth. Rev. Bras. Farmacogn. 16, 73–76. doi:
10.1590/S0102-695X2006000100013
Felix, I., Reinhard, S., and Gudrun, A. J. R. (2009). Additive Antmicrobial Effects of
the Active Components of the Essential Oil of Thymus vulgaris - Chemotype
Carvacrol. Planta Med. 75, 1231–1236. doi: 10.1055/s-0029-1185541
Chemical-Computing-Group-ULC (2018). Molecular Operating Environment
(MOE), 2013.08 (Montreal, QC, Canada). Available at: www.chemcomp.com.
Chen, Y., Zhang, H., Zhou, H. J., Ji, W., and Min, W. (2016). Mitochondrial Redox
Signaling and Tumor Progression. Cancers (Basel). 8, 1–15. doi: 10.3390/
cancers8040040
Frontiers in Pharmacology | www.frontiersin.org
10
July 2020 | Volume 11 | Article 1125

´
Valdes et al.
Flavanones From Calceolaria thyrsiflora
Fischer, W., Currais, A., Liang, Z., Pinto, A., and Maher, P. (2019). Old age-
associated phenotypic screening for Alzheimer’s disease drug candidates
identifies sterubin as a potent neuroprotective compound from Yerba santa.
Redox Biol. 21, 1–12. doi: 10.1016/j.redox.2018.101089
Molau, U. (1988). Scrophulariaceae Part I. Calceolarieae. Flora Neotrop. Vol. 47,
(The New York Botanical Garden Press), pp. 325.
Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell,
D. S., et al. (2009). AutoDock4 and AutoDockTools4: Automated Docking with
Selective Receptor Flexibility. J. Comput. Chem. 30, 2785–2791. doi: 10.1002/
jcc.21256
Mossa, J. S., Sattar, E. A., and Galal, A. M. (1996). Free Flavonoids from Rhus
retinorrhoea Steud, ex Olive. Int. J. Pharmacogn. 34, 198–201. doi: 10.1076/
phbi.34.3.198.13206
Muñoz, M. A., Bucio, M. A., and Joseph-Nathan, P. (2013). Chiroptical Studies of
Flavanone. Nat. Prod. Commun. 8, 1075–1078. doi: 10.1177/1934578X1300800810
NCCLS (1999). “Methods for Determining Bactericidal Activity of Antimicrobial
Agents; Approved Guideline,” in NCCLS document M26-A (940 West Valley
Road, Suite 1400, Wayne, Pennsylvania 19087 USA: NCCLS), ISBN: .
Ndoile, M. M., and Van Heerden, F. R. (2013). Total synthesis of ochnaflavone.
Beilstein J. Org. Chem. 9, 1346–1351. doi: 10.3762/bjoc.9.152
Steinhilber, D., Fischer, A. S., Metzner, J., Steinbrink, S. D., Roos, J., Ruthardt, M.,
et al. (2010). 5-Lipoxygenase: underappreciated role of a pro-inflammatory
enzyme in tumorigenesis. Front. Pharmacol. 1, 143. doi: 10.3389/
fphar.2010.00143
Sun, J., Zhou, W., Wei, C. X., Zhang, Z., Jin, X., and Li, G. (2019). A new
benzofuran from Artemisia halodendron Turcz. ex Bess. Nat. Prod. Res. 33,
226–232. doi: 10.1080/14786419.2018.1443098
Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A.,
Cheeseman, J. R., et al. (2016). Gaussian 03. (Wallingford, CT: Gaussian, Inc.).
Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Rob, M. A., Cheeseman, J. R.,
et al. (2003). Gaussian 09, Revision A.02, Gaussian Inc (CT, USA: Wallingford).
Garbarino, J. A., Cardile, V., Lombardo, L., Chamy, M. C., Piovano, M., and Russo,
A. (2007). Demalonyl thyrsiflorin A, a semisynthetic labdane-derived
diterpenoid, induces apoptosis and necrosis in human epithelial cancer cells.
Chem. Biol. Interact. 169, 198–206. doi: 10.1016/j.cbi.2007.06.031
Ghosh, J., and Myers, C. E. (1998). Inhibition of arachidonate 5-lipoxygenase
triggers massive apoptosis in human prostate cancer cells. Proc. Natl. Acad. Sci.
U. S. A. 95, 13182–13187. doi: 10.1073/pnas.95.22.13182
GraphPad-Software (2015). GraphPad Prism version 6.00 for Windows. La Jolla,
California, USA: GraphPad Software. Available at: www.graphpad.com.
GraphPad-Software-Inc (2017). Prism version Demo 7. La Jolla, California, USA:
GraphPad Software. Available at: www.graphpad.com.
Grotewold, E. (2006). The Science of Flavonoids. Ed. E. Grotewold (New York, NY:
Springer New York).
Horowitz, R., and Gentili, B. (1961). Phenolic Glycosides of Grapefruit: a Relation
between Bitterness and Structure. Arch. Biochem. Biophys. 92, 191–192. doi:
10.1016/0003-9861(61)90235-1
́
Taborga, L., Diaz, K., Olea, A., Reyes-Bravo, P., Flores, M., Peña-Cortes, H., et al.
Hyun, J., Eun, Y., Sohn, J., and Hyun, D. (2010). A facile method for the rapid and
selective deprotection of methoxymethyl (MOM) ethers. Tetrahedron 66,
1673–1677. doi: 10.1016/j.tet.2010.01.007
(2015). Effect of polymer micelles on antifungal activity of geranylorcinol
compounds against Botrytis cinerea. J. Agric. Food Chem. 63, 6890–6896. doi:
10.1021/acs.jafc.5b01920
Jerz, G., Waibel, R., and Achenbach, H. (2005). Cyclohexanoid protoflavanones
from the stem-bark and roots of Ongokea gore. Phytochemistry 66, 1698–1706.
doi: 10.1016/j.phytochem.2005.04.031
Vasconcelos, J. M. J., Silva, A. M. S., and Cavaleiro, J. A. S. (1998). Chromones and
flavanones from Artemisia campestris subsp. Maritima. Phytochemistry 49,
1421–1424. doi: 10.1016/S0031-9422(98)00180-0
́
́
́
Joseph-Nathan, P., Giordano, O. S., and Romo, J. (2015). “Vibrational circular
dichroism absolute configuration determination of natural products,” in
Progress in the Chemistry of Organic Natural Products. Eds. A. D. Kinghorn,
H. Falk and J. Kobayashi (Switzerland: Cham), 311–415.
Vasquez-Martınez, Y., Osorio, M. E., San Martın, D., Carvajal, M. A., Vergara,
A. P., Sanchez, E., et al. (2019). Antimicrobial, Anti-Inflammatory and
Antioxidant Activities of Polyoxygenated Chalcones. J. Braz. Chem. Soc 30,
286–304. doi: 10.21577/0103-5053.20180177
Kavka, J., Guerreiro, E., and Giordano, O. J. R. (1973). Flavonoids of Baccharis
salicifolia. Rev. Latinoam. Quim. 4 (3), 101–104.
Ketabforoosh, S. H. M. E., Kheirollahi, A., Safavi, M., and Esmati, N. (2014).
Synthesis and Anti-Cancer Activity Evaluation of New Dimethoxylated
Chalcone and Flavanone Analogs. Arch. Pharm. Chem. Life Sci. 347, 853–
860. doi: 10.1002/ardp.201400215
Villagran, C., and Hinojosa, L. F. (1997). Historia de los bosques del sur de
́
́
́
Sudamerica, II: Analisis fitogeografico. Rev. Chil. Hist. Nat. 70, 241–267.
Wavefunction-Inc (2004). Spartan version 04 Softwre package. Irvine, California,
USA. Available at: www.wavefun.com.
Wurm, G., and Geres, U. (1982). Eine neue Eriodictyolsynthese. Arch. Pharm.
(Weinheim). 315, 183–185. doi: 10.1002/ardp.19823150217
Khupse, R. S., and Erhardt, P. W. (2007). Total Synthesis of Xanthohumol. J. Nat.
Prod. 70, 1507–1509. doi: 10.1021/np070158y
Ley, J. P., Krammer, G., Reinders, G., Gatfield, I. L., and Bertram, H. J. (2005).
Evaluation of Bitter Masking Flavanones from Herba Santa (Eriodictyon
californicum (H. & A.) Torr., Hydrophyllaceae). J. Agric. Food Chem. 53,
6061–6066. doi: 10.1021/jf0505170
Zhao, D.-H., Sui, X., Qu, Y.-L., Yang, L.-Y., Wang, X., and Guan, L.-P. (2011).
Synthesis and Studies on Antidepressant Effect of 5,7-Dihydroxyflavanone
Derivatives. Asian J. Chem. 23 (3), 1129–1132.
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
́
́
́
Madrid, A., Dıaz, K., Gonzalez, C., Catalan, K., and Espinoza, L. (2015).
Antiphytopathogenic activity of Psoralea glandulosa (Fabaceae) against
Botrytis cinerea and Phytophthora cinnamomi. Nat. Prod. Res. 29, 586–588.
doi: 10.1080/14786419.2014.955486
́
́
́
́
́
Copyright © 2020 Valdes, Gonzalez, Dıaz, Vasquez-Martınez, Mascayano, Torrent,
́
Cabezas, Mejias, Montoya, Cortez-San Martın, Muñoz, Joseph-Nathan, Osorio and
Ministerio de Salud de Chile (2010). Medicamentos Herbarios Tradicionales.
Available at: http://web.minsal.cl/sites/default/files/files/Libro MHT 2010.pdf
(Accessed January 18, 2017).
Mitra, S., Bartlett, S. G., and Newcomer, M. E. (2015). Identification of the
substrate access portal of 5-Lipoxygenase. Biochemistry 54, 6333–6342. doi:
10.1021/acs.biochem.5b00930
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