Study of two isoforms of lipoxygenase by kinetic assays, docking and molecular dynamics of a specialised metabolite isolated from the aerial portion of Lithrea caustica (Anacardiaceae) and its synthetic analogs

Article abstract of DOI:10.1016/j.phytochem.2020.112359

Our investigation focused on the characterization and study of epicuticular leaf extracts (dichloromethane extract) and certain derivatives of Lithrea caustica (Molina) Hook and Arn. (Anacardiaceae) as inhibitors of 15 soybean and 5 human lipoxygenases (1

Full text of DOI:10.1016/j.phytochem.2020.112359

Phytochemistry 174 (2020) 112359
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Study of two isoforms of lipoxygenase by kinetic assays, docking and
molecular dynamics of a specialised metabolite isolated from the aerial
portion of Lithrea caustica (Anacardiaceae) and its synthetic analogs
∗
∗
∗
Alejandra Muñoz-Ramírez , Claudia Torrent-Farías, Carolina Mascayano-Collado ,
Alejandro Urzúa-Moll
Departamento de Ciencias Del Ambiente, Facultad de Química y Biología, Universidad de Santiago, Chile, Casilla 442, Correo 2, Santiago, Chile
A R T I C L E I N F O
A B S T R A C T
Keywords:
Our investigation focused on the characterization and study of epicuticular leaf extracts (dichloromethane ex-
tract) and certain derivatives of Lithrea caustica (Molina) Hook and Arn. (Anacardiaceae) as inhibitors of 15
soybean and 5 human lipoxygenases (15-sLOX and 5-hLOX). From the epicuticular extract of leaves, the com-
pound (Z)-3-(pentadec-10′-enyl)-catechol (Litreol) was isolated, and three hemisynthetic derivatives were pre-
pared, as they are 3-pentadecylcatechol, (Z)-1,2-diacetyl-3-(pentadec-10′-enyl)-benzene and 1,2-diacetyl-3-
pentadecylbenzene. The inhibitory activities for the four compounds against 15-sLOX and 5-hLOX were de-
termined, being (Z)-3-(pentadec-10′-enyl)-catechol (IC50 54.77 μM and 2.09 μM, respectively) and 3-pentade-
cylcatechol (IC50 55.28 μM and 2.74 μM, respectively), the most interesting compounds assayed. The kinetic
studies for (Z)-3-(pentadec-10′-enyl)-catechol and 3-pentadecylcatechol showed a mixed inhibition mechanism
to 5-LOX. Finally, docking and molecular dynamics studies were performed to characterize and describe how the
chemical structures could be correlated to the decreased 5-hLOX activity observed in the in vitro studies.
Lithrea caustica
Anacardiaceae
Natural products
5
-LOX inhibition
Anticancer agents
Molecular dynamics
1. Introduction
dermatitis, also contain anti-inflammatory compounds. These species
include Mangifera indica L. (mango) (Garro et al., 2015) and species of
The Anacardiaceae family consists of approximately 68 genera and
00 species, among which are trees, shrubs and lianas of wide dis-
the genus Lithraea (Alé et al., 1997).
6
The genus Lithraea is represented by four species distributed across
Australia and the Americas. Three South American species (L. caustica,
L. molleoides and L. brasiliensis) are known to produce allergic contact
dermatitis (ACD) (Gambaro et al., 1986; López et al., 1998).
L. caustica (Molina) Hook. And Arn. (Anacardiaceae), commonly
known as “litre”, is an evergreen tree, shrub or creeping plant that
ranges from 0.5 to 3 m tall, is endemic to Chile (Huang et al., 2018;
Teillier et al., 2018).
A study of the stem bark and leaves of L. caustica was carried out,
and an alkylcatechol was isolated, commonly called “litreol”, whose
structure was determined as 3-(pentadec-10′-enyl)-catechol (Gambaro
et al., 1986) and is associated with the allergy produced by the litre
(López et al., 1998).
tribution from mostly tropical climates (Perrotta and Arambarri, 2004).
Some species of Anacardiaceae are recognized for synthesizing a
characteristic group of compounds, shown varied biological properties,
including allergenic, antibacterial, fungicidal, antitumor, anti-in-
flammatory activities, etc. (Russo et al., 2009; Diby et al., 2016). These
compounds constitute a family of lipophilic molecules that share a
general structure, a catechol group at position 3 is substituted with one
chain of 10, 12, 14, 15 or 17 carbon atoms, with one or more positions
of unsaturation in the main chain, forming a complex mixture of al-
kylcatechols with very similar physicochemical properties (Kalergis
et al., 1997; Aziz et al., 2017). For example, “urushiol”, an alkylca-
techol (see Fig. 1), are a group of alkyls catechol that differing in the
length and number of unsaturations of their alkyl chain (Gambaro et al.,
In 2011, the study of epicuticular components of two populations of
L. caustica by extracting fresh leaves using dichloromethane was per-
formed. The extracts exhibited a mixture of n-alkanes of 21–33 carbon
units as their main components and small levels of monoterpene
1
986) and are known to cause irritation, inflammation and blisters on
the skin (Huang et al., 2018).
Some species of the Anacardiaceae family, which cause contact
∗
Corresponding author.
∗∗
Corresponding author.
E-mail addresses: alejandra.munozr@usach.cl (A. Muñoz-Ramírez), carolina.mascayano@usach.cl (C. Mascayano-Collado).
https://doi.org/10.1016/j.phytochem.2020.112359
Received 23 May 2019; Received in revised form 17 March 2020; Accepted 18 March 2020
0031-9422/ © 2020 Elsevier Ltd. All rights reserved.

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
Fig. 1. Basic structure of urushiol.
Fig. 2. Formation of tropylium ion with two phenolic –OH groups.
hydrocarbons in addition to the presence of 3-(pentadec-10′-enyl)-ca-
techol, demonstrating that it is found in the epicuticle (Urzúa et al.,
silica gel chromatography. Pure fractions after chromatography showed
an intense dark blue spot when we used 10% FeCl3, indicating the
presence of phenols. Finally, these fractions were regrouped and crys-
tallized in methanol, yielding 0.342 g of a white solid and 0.193 g of an
oil corresponding to the phenolic fraction, both of which were mon-
itored by TLC and analyzed using 10% FeCl3.
To determine the complexity, proportion and purity of the epicuti-
cular extract of the two products obtained, we used gas chromato-
graphy (GC) analysis. We observed a series of minor peaks between 10
and 30 min, but after 50 min, the most important signal appeared with
purity of 90% (shown in Supplementary data).
2011).
In folk medicine, the tincture of leaves is applied at homeopathic
doses for squamous diseases of the skin, the leaves are eaten raw to
avoid allergy problems (San Martín, 1983) and the stem juice of L.
caustica with Rubus ulmifolius (blackberry) is used to treat coughs
(
Russo et al., 2009). Additionally, in Mapuche medicine, an infusion of
leaves is used for parts of the body presenting with articular in-
flammations, which are submerged in the infusion (Gusinde, 1936;
Montecino and Conejeros, 1985).
Inflammation protects the body against infections and injuries, but
the response can cause various diseases, such as rheumatoid arthritis
and inflammation of the intestine and psoriasis, among others.
LOXs regulate the pathophysiology of various diseases (Yar et al.,
With respect to GC of the solid, three major peaks were identified
that were similar to the major peaks present in the epicuticular extract,
showing a mixture of compounds of different molecular weights. On the
other hand, the phenolic fraction exhibited a large signal at 52 min.
2014), also play an essential role in the biosynthesis of lipoxins (LX),
thromboxanes (TX) and leukotrienes (LT) that generate the physiolo-
gically active oxygenated fatty acids (Nikolaev et al., 1990; Li et al.,
2.2. Chemistry
2
015; Imai et al., 2016).
The enzyme 5-lipoxygenase (5-LOX), one of the most physiologi-
Characterization of the phenolic fraction was performed using
spectroscopic techniques, such as IR-FT, GC-MS, 1H and 13C NMR
shown in Supplementary data). The IR-FT spectrum revealed a wide
signal centered at 3441 cm-1, which was attributed to the HO stretching
of hydroxyl groups, and a band was observed at the lower frequency of
004 cm-1, which was attributed to the CH stretching of Csp 2-H bonds.
The series of signals centered on 2953 cm-1, 2923 cm-1 and 2852 cm-1
were assigned to the C–H stretching of aliphatic groups. Finally, at a
lower frequency, 1463 and 1277 cm-1 bands appeared, which were
attributed to the C–O stretch present in the ring.
The chromatogram of the GC-MS analysis of the phenolic fraction
showed a major peak at 38.5 min. The mass spectrum showed an M+ at
cally important lipoxygenases (Giribaldi et al., 1993; Chen et al., 1998),
produces LTs, that are formed during the decomposition of arachidonic
acid (AA) through the LOX pathway (Koshihara et al., 1983; Shimizu
et al., 1984; Pontiki et al., 2011), and have been implicated in the
pathogenesis of disorders associated with inflammation, for example,
asthma and allergic rhinitis, and have become major targets for ther-
apeutic modulation (Busse, 1996).
The development of new and safe anti-inflammatory agents remains
a subject of great interest (Gorzalczany et al., 2011). A group within the
candidates for this type of research are specialised metabolites of plants
used in folk medicine to treat inflammatory processes.
(
3
3
18 units of atomic mass (uma) consistent with formula C21H34O2 that
has five unsaturated hydrocarbons. The base peak at m/z 123 (C
suggested a tropylium cation with two phenolic –OH groups (Fig. 2).
In this context, the present investigation focused in the chemical
study of L. caustica, with the purpose of evaluating the activity of its
specialised metabolites as inhibitors of 5-hLOX and 15-sLOX and cor-
relating those results with in silico studies.
7 7 2
H O )
By GC-MS analysis, an alkylcatechol or an alkylresorcinol with an
unsaturated hydrocarbon chain of 15 carbon atoms was predicted. The
1H and 13C NMR analysis of the phenolic fraction obtained from the
2
. Results and discussion
epicuticular extract of L. caustica showed that the major compound
corresponded to (Z)-3-(pentadec-10′-enyl)-catechol (1) (Fig. 3). From
(Z)-3-(pentadec-10′-enyl)-catechol three synthetic derivatives: 3-pen-
tadecylcatechol, 1,2-diacetyl-3-pentadecylbenzene, (Z)-1,2-diacetyl-3-
2.1. Purification and analysis of epicuticular extract (dichloromethane
extract)
(
pentadec-10′-enyl)-benzene, were synthetized (See Supplementary
The epicuticular extract of L. caustica (2.51 g), was fractionated by
data).
2

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
Fig. 3. (Z)-3-(pentadec-10′-enyl)-catechol structure.
2.3. 5- and 15-LOX inhibition, antioxidant and iron-reducing capacity, and
Table 1
enzymatic studies and docking
Percent inhibition of the enzyme 5-hLOX with respect to antioxidant and re-
ductive capacity as measured by the DPPH, ABTS and FRAP methods. Values of
m i
maximum speed (Vmax), Michaelis constant (K ) and inhibition constant (K )
are also shown for (Z)-3-(pentadec-10′-enyl)-catechol (1) and 3-pentadecylca-
techol (2).
2
.3.1. Inhibition of 15- and 5-LOX activity
Examination of the inhibitory activity of (Z)-3-(pentadec-10′-enyl)-
catechol (1) against 5-hLOX was carried out using nordihydroguaiaretic
acid (NDGA) as a positive control. The procedure was carried out by
fluorimetry (Singh et al., 2012) as described in more detail later in this
section. For the determination of the IC50, different concentrations of
the inhibitor were prepared using DMSO as the solvent. Biological
studies showed a good and promissory IC50 (2.09 μM), but not com-
parable to good and known 5-hLOX inhibitors (Singh et al., 2012), such
as zileuton, atreleuton and NDGA, with IC50 values between 0.32, 0.08
and 0.02 μM, respectively. Furthermore, zileuton, an iron chelator (Nair
and Funk, 2009), is the only drug approved by the Food and Drug
Administration (FDA) for psoriasis treatment.
To determine whether (Z)-3-(pentadec-10′-enyl)-catechol (1) is a
specific inhibitor of 5-hLOX, we decided to examine with other LOX
isoform, 15-sLOX (shown in Supplementary data). The 15-sLOX enzyme
was taken as a study model because, although the human enzyme is also
commercially available, it is less stable than that of plant origin, ex-
hibiting low biological activity by in vitro studies. The alignment of the
sequences of human and soybean 15-LOX yielded a 28% identity. Al-
though this could be taken as an indication of little identity between the
two isoforms, a comparative study of their active sites showed that all
residues that are part of the binding site, and that interact with the AA
and iron (Wecksler et al., 2009), are highly conserved in both systems,
being the 15-sLOX a first good model for studying human 15-LOX in-
hibitors.
Screening inhibitory activity of 5-hLOX
(
1)
69.6
3.7
(2)
(3)
NA
NA
NA
NA
–
(4)
NA
NA
NA
NA
–
NDGA
96.7
95.1
88.7
93.5
%
Inhibition
79.4
76.3
73.9
76.6
± 2.7
7
63.1
Inhibition average
SD
68.8
± 5.3
± 4.2
Antioxidant and iron-reducing capacity
Assays
DPPH
(1)
(2)
(3)
(4)
Background
% free radicals
entrapment
% free radicals
entrapment
Antioxidant
capacity/Iron-
reducing (μg/
mL)
16.7
83.3
10.2
89.8
33.61
16.2
83.8
10.1
89.9
90.0 89.5 100
9.9 10.5
75.1 74.4 100
%
0
ABTS
FRAP
%
24.9 25.6
0
–
45.34 4.84 4.34
Enzymatic kinetics
Inhibitor
(2)
(1)
0
[I] μM
max
0
0.5
1242
73.6
1.5
3
0.5
1.5
3
V
1949
36.9
1042 944.7 1435
87.3 111 33.8
1280 1025 794
78.3 84.8 95.9
Km
K
i app (μM) 0.33 (Mixed Mechanism)
(0.14)
Ki’ (0.71)
0.95 (Mixed Mechanism)
(0.40)
Ki’ (1.95)
K
i
K
i
The IC50 value obtained for 15-sLOX was 54.77 μM, which is less
potent than previously reported inhibitors, such as baicalein (35 μM) or
kaempferol (50 μM) (Sadik et al., 2003). When we compared the IC50
values obtained for both enzymes, 5-hLOX (2.09 μM) and 15-sLOX
other types of systems in previous research (Sugiura et al., 1989;
Schaible et al., 2016). These differences could be due to different in-
hibitory mechanisms between LOX isoforms.
(
54.77 μM), the (Z)-3-(pentadec-10′-enyl)-catechol (1) showed to be a
potent and selective inhibitor of 5-hLOX compared to 15-sLOX. One
possible explanation for this discrepancy may be differences in the size
of the active sites for both enzymes, as the 5-hLOX active site is 20%
larger than the 15-sLOX active site (Gillmor et al., 1997) and the their
analysis of sizes of cavities in both enzymes, allowing give the struc-
tural explications about as the 5th or 15th carbons of the arachidonic
acid are placed in front to metal and beginning the catalytic cycle.
Analysis of the (Z)-3-(pentadec-10′-enyl)-catechol (1) structure and
its relationship to 5-LOX inhibition revealed two keys factors: the ca-
techol group, that would affect the potency against 5-LOX and the alkyl
chain (Peduto et al., 2017). For this, three derivatives were synthesized,
two of which block the catechol group through acetylation as com-
pounds (3) and (4) and one with saturated side chain (2). Table 1 shows
the average values of 5-hLOX activities in the presence of the different
synthetic derivatives. The main results showed that blocking the ca-
techol group of (Z)-3-(pentadec-10′-enyl)-catechol (1) using acetyl
groups dramatically decreased inhibition, but this difference was not
observed when we reduced the double bond through catalytic hydro-
genation. In parallel, we made the same comparison but with 15-sLOX.
The IC50 showed that (Z)-3-(pentadec-10′-enyl)-catechol (1) (54.77 μM)
and 3-pentadecylcatechol (2) (55.28 μM) did not exhibit dramatic de-
creases of inhibition whether we used the unsaturated chain or not,
unlike what we observed in 5-hLOX, as was demonstrated comparing
2
.3.2. Antioxidant activity
Due to the results described above and the importance of catechol to
-hLOX inhibition, three different methods to estimate antioxidant ac-
5
tivity were performed. DPPH evaluates antioxidant activity using the
free radical 2,2-diphenyl-1-picrylhydracil (DPPH), which determines
radical capture in the presence of an antioxidant substance by spec-
trophotometry at 520 nm. ABTS is based on quantification of the dis-
coloration of the radical ABTS • + due to interaction with donor species
of hydrogens or electrons. The cationic radical ABTS • + is a chromo-
phore that absorbs at 734 nm and is generated by an oxidation reaction
of ABTS (2,2′-azino-bis-(3-ethyl benzthiazolin-6-sulfonate ammonium)
with potassium persulfate. In contrast, FRAP measures the antioxidant
capacity of a sample based on its ability to reduce the ferric iron Fe (III)
present in 2,4,6-tri-(2-pyridyl)-s-triazine (TPTZ) to the ferrous form Fe
(
II) and has a maximum absorbance at a wavelength between 590 and
5
95 nm. The results are shown in Table 1.
Table 1 shows that the compounds with the highest antioxidant
capacity, according to the DPPH and ABTS assays, were 3-pentade-
cylcatechol (2) and (Z)-3-(pentadec-10′-enyl)-catechol (1), which were
the two compounds with catechol groups in their structure. Replacing
the catechol groups with diacetylated derivatives (3) and (4) decreased
the antioxidant capacity considerably. The FRAP experiments
3

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
Fig. 4. (Z)-3-(pentadec-10′-enyl)-catechol (1) and 3-pentadecylcatechol (2) Lineweaver-Burk graphs.
demonstrated the same behavior, and compounds that had greater
antioxidant properties or iron-reducing capacities were the compounds
with a catechol group in their structure. These results are in agreement
with the IC50 experiments described above.
and (2) and to correlate our biological and antioxidant experiments, we
carried out molecular dynamics simulations (MD) with 5-hLOX. The
analysis of MD simulations showed that the compound (2) interacted
with the residue ASN-407 through donor hydrogen bonding.
Furthermore, we observed that the catechol group initially tended to
move away from the metallic center, with an average maximum dis-
tance of 4.3 Å and a minimum at 2.8 Å at 10 ns of MD. The same
behavior was observed with the compound (1), and the main interac-
tion found was with ASN-407 as we showed with (2), with the
minimum average distance at 3.7 Å.
Following our MD study and the mixed mechanism determined by
kinetic experiments, we next performed MD using the substrate ara-
chidonic acid (AA) and the best inhibitors, (1) and (2) found. The re-
sults demonstrated that in the first system (Complex 2-AA), the mo-
lecules were positioned near the binding site, with the principal
interaction occurring between the derivate (2) and ASN-407, which
placed the catechol group very close to the metal. Meanwhile, AA in-
teracted with THR-364, HIS-432, GLN-557, GLN-363 and THR-427, as
has been previously described in the literature (Gillmor et al., 1997).
Fig. 5A shows the distance between the catechol group and the iron
with a change along the simulation time. In an initial state, the average
distance was 6.8 Å, but during and at the end of the MD we observed
that the catechol slowly approached at a distance of 5.4 Å, and AA
placed C-7 of the hydrocarbon chain in front of the metal during the
initial state of MD. These results show a slight preference to the enzyme
for the substrate compared with the derivate (2). Similar simulations
were done by MD, between AA and the molecule (1) (Fig. 5B), the
fluctuation of the distance between the catechol group and iron was
2.3.3. Kinetic experiments
Based on our results, we next investigated the type of inhibition by
(
Z)-3-(pentadec-10′-enyl)-catechol (1) and 3-pentadecylcatechol (2)
against 5-hLOX using enzyme kinetics. The enzyme 5-hLOX has iron as
a central atom, and when in the ferric state Fe (III), the enzyme is
catalytically active, while in its ferrous state Fe (II), the enzyme is in-
active. To determine the mechanism of inhibition by (1) and (2) against
5
-hLOX, an examination of the enzymatic kinetics was carried out to
determine the binding characteristics of these compounds with the
enzyme. The appropriate kinetics parameters as V , K and K were
m
m
i
obtained directly from v and s values without any transformation to a
reciprocal by Michaelis-Menten. The representation of the respective
inhibition mechanisms were obtained for (1) and (2) by kinetics ex-
periments as we shown in Fig. 4. In both cases, the same behaviors were
m m
observed, a decrease V and a change in K , as results the Ki app, were
0
.95 and 0.33 μM respectively, suggesting a mixed mechanism of in-
hibition in both compounds. These results represent that the inhibitor
can bind to the free enzyme (competitive) and to the ES complex (un-
competitive). The analysis to the K
the compound (1) had a higher preference for competitive mechanism
) unlike to the uncompetitive preference of (2) (Km,
m
, app in (1) and (2), indicated that
(
K
m, app > K
m
app < K
m
).
4
.4 Å at 3.5 Å, indicating the higher affinity to the inhibitor than the
substrate to 5-hLOX. Finally, both results obtained for the compounds
1) and (2) by MD they were agree with the mixed mechanism obtained
2
.3.4. Docking
We next performed in silico studies to understand the binding mode
(
for the two most potent and selective inhibitors. The first study loca-
lized the binding site of ligands by docking using the crystal structure of
by enzymatic assays.
5-hLOX (PDB ID 3O8Y) at a resolution of 2.30 Å. The results revealed
that all ligands were in the active site but exhibited differential affi-
nities. For example, the ΔG value obtained for the ligand (1)
3. Conclusions
(
(
−5.55 kcal/mol) was comparable in affinity and energy to (2)
−5.07 Kcal/mol), demonstrating two principal interactions with the
The (Z)-3-(pentadec-10′-enyl)-catechol (1) was evaluated for the
first time against LOX isoforms, revealing significant inhibition of en-
zymatic activity for two enzymes (15-sLOX, IC50 54.77 μM; 5-hLOX,
IC50 2.09 μM), demonstrating that it is a relatively specific inhibitor of
5-hLOX. In addition, the enzymatic activity of the reduction product of
(Z)-3-(pentadec-10′-enyl)-catechol (1), 3-pentadecylcatechol (2), and of
the respective diacetylated derivatives, (Z)-1,2-diacetyl-3-(pentadec-
10′-enyl)-benzene (3) and 1,2-diacetyl-3-pentadecylbenzene (4), were
determined, demonstrating that the presence of a catechol group in this
type of system is essential for the inhibition of 5-hLOX and not the
unsaturated chains.
residues ILE-637 and ASN-407 by hydrogen bonding with the catechol
group oriented in front of the iron at a distance of approximately 2.5 Å.
Finally, the binding energies detected for the acetylated derivatives (3)
and (4) were +7.66 Kcal/mol and +7.78 Kcal/mol, respectively.
We observed that (1) and (2) oriented the catechol group towards
the metallic center. This characteristic, together with the results ob-
tained from the iron reducing capacity (FRAP), DPPH and ABTS in
Table 1, suggest a redox inhibition that comprises a mixed mechanism
identified by enzyme kinetics.
Through enzyme kinetics studies, it was determined that (Z)-3-
(pentadec-10′-enyl)-catechol (1) and 3-pentadecylcatechol (2) were
potent and selective inhibitors the 5-hLOX and a mixed inhibition
2
.3.5. Molecular dynamics
To determine the importance of the catechol groups, present in (1)
4

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
Fig. 5. Active site of molecular dynamics between 3-pentadecylcatechol (2) (A), (Z)-3-(pentadec-10′-enyl)-catechol (1) (B), and arachidonic acid with 5-hLOX and
fluctuation of catechol distances during simulation time (10 ns).
mechanism with a K
1) and 0.33 μM for 3-pentadecylcatechol (2) were obtained, which was
confirmed by MD simulations.
i
of 0.95 μM for (Z)-3-(pentadec-10′-enyl)-catechol
4.3. Procedure for synthesis of 3-pentadecylcatechol (2)
(
To a solution of (Z)-3-(pentadec-10′-enyl)-catechol (1) (0.88 mmol)
in 20 mL methanol, 10% catalyst Pd/C was added. The reaction was
carried out in a reactor at 2 bar of hydrogen pressure stirring for 4 h at
room temperature. The product was then filtered and washed with
methanol. Physical and NMR data are listed below:
4. Experimental
4.1. Plant material
4
.3.1. (Z)-3-(pentadec-10′-enyl)-catechol (1)
Yield 8%; oil. 1H NMR (CDCl3, 400 MHz, δ, ppm) δ: 6.71 (s, 3H,
Leaves of Lithrea caustica (Molina) Hook and Arn. (Anacardiaceae)
were collected during the flowering season, Abril 2017, in Farellones,
Santiago Metropolitan Region, Chile (33° 18′ 35.9″S; 70° 19′ 19.9″W) at
altitudes of 1200–1300 m above sea level (masl). Voucher specimens
SGO-067800) were deposited in the Herbarium of the National
Museum of Natural History, Santiago, Chile.
CH); 5.36 (t, 2H, JH-H = 4.5 Hz, CH); 2.60 (t, 2H, JH-H = 7.6 Hz,
CH2); 2.02 (m, 2H, CH2); 1.40–1.26 (m, 18H, CH2); 0.90 (t, 3H, JH-
H = 6.6, CH3). 13C NMR (CDCl3, 400 MHz, δ, ppm) δ: 143.0 (s, C);
1
1
2
(
41.9 (s, C); 129.9 (s, CH); 129.8 (s, CH); 129.3 (s, C); 122.0 (s, CH);
20.0 (s, CH); 112.8 (s, CH); 31.9 (s, CH2); 29.8–29.4 (m, 7C, CH2);
9.3 (s, CH2); 27.2 (s, CH2); 26.9 (s, CH2); 22.3 (s, CH2); 14.0 (s, CH3).
4
.1.1. Extraction of epicuticular resin with dichloromethane. Epicuticular
extract
Fresh leaves of L. caustica (1.4 kg) were placed in 1 L beaker and
4.3.2. 3-Pentadecylcatechol (2)
Yield 62%; solid, 1H NMR (CDCl3, 400 MHz, δ, ppm) δ: 6.71 (s, 3H,
CH); 2.60 (t, 2H, JH-H = 7.8 Hz, CH2); 1.26 (m, 26H, CH2); 0.89 (t,
3H, JH-H = 6.5, CH3). 13C NMR (CDCl3, 400 MHz, δ, ppm) δ: 143.5 (s,
C); 142.4 (s, C); 129.8 (s, C); 122.4 (s, CH); 120.4 (s, CH); 113.2 (s, CH);
32.3 (s, CH2); 30.2–29.8 (m, 12C, CH2); 23.1 (s, CH2); 14.5 (s, CH3).
dichloromethane was added to cover the volume of the leaves (final
volume 5 L), which were macerated for 5 min by stirring with a glass
rod. Next, the solution was filtered, and the dichloromethane was
eliminated under reduced pressure in a rotary evaporator. Finally, the
extract was stored in the dark at 5 °C, yielding (6.1 g, 0.44%).
4.4. General procedure for synthesis of diacetylated derivatives
4
4
.2. Chemistry
2 2
To a solution of alkyl catechol (0.19 mmol) in 8 mL CH Cl ,
.22 mmol acetic anhydride and 4-dimethylaminopyridine (0.20 mmol)
2
.2.1. Gas-liquid chromatography analysis of epicuticular extract and
were added. The mixture was stirred for 24 h at room temperature.
Next, the solution was washed with 5% HCl, followed by 6% NaHCO
isolated compounds
3
The epicuticular extract and isolated compounds from L. caustica
were analyzed by GLC using a Shimadzu GC-2010 Plus equipped with
an HP-5 column (30 m × 0.250 mm x 0.25 μm), samples (10 mg/mL)
were prepared in dichloromethane. The chromatogram was developed
using a thermal gradient as follows: 5 min at 40 °C, 10 °C/min to 200 °C,
and finally with water. After washing, the solution was dried using
anhydrous sodium sulfate, then filtered and evaporated. Physical and
NMR data are listed below:
4
.4.1. (Z)-1,2-diacethyl-3-(pentadec-10′-enyl)-benzene (3)
Yield 71.8%; oil. 1H NMR (CDCl3, 400 MHz, δ, ppm) δ: 7.86 (d, 1H,
5
min at 200 °C; 3 °C/min to 300 °C, 20 min at 300 °C.
JH-H = 7.5 Hz, CH); 7.68 (t, 1H, JH-H = 8.2 Hz, CH); 7.31 (d, 1H, JH-
H = 8.2 Hz, CH); 5.35 (t, 2H, JH-H = 4.7 Hz, CH); 2.51 (t, 2H, JH-
H = 7.6 Hz, CH2); 2.31 (s, 3H, CH3); 2.27 (s, 3H, CH3); 2.02 (m, 2H,
CH2); 2.01 (m, 2H, CH2); 1.33–1.26 (m, 18H, CH2). 13C NMR (CDCl3,
400 MHz, δ, ppm) δ: 168.7 (s, C=O); 168.6 (s, C=O); 142.8 (s, C);
140.9 (s, C); 137.1 (s, C); 130.2 (s, CH); 129.9 (s, CH); 127.6 (s, CH);
126.5 (s, CH); 121.2 (s, CH); 32.3 (s, CH2); 31.9 (s, CH2); 30.4–29.6 (m,
.2.2. IR-FT, H and 13C NMR analysis
1
4
The IR-FT spectra of (Z)-3-(pentadec-10′-enyl)-catechol (1), 3-pen-
tadecylcatechol (2), (Z)-1,2-diacetyl-3-(pentadec-10′-enyl)-benzene (3)
and 1,2-diacetyl-3-pentadecylbenzene (4) were imaged on film using a
PerkinElmer Spectrum 65, and H and C NMR spectra were recorded
on a Bruker Advance 400 (400 MHz) (shown in Supplementary data).
1
13
5

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
7
2
C, CH2); 27.5 (s, CH2); 27.3 (s, CH2); 22.7 (s, CH2); 21.1 (s, CH3);
0.7 (s, CH3); 14.3 (s, CH3).
4.8. Evaluation of radical-trapping activity using ABTS
To prepare the reaction, sodium persulfate and ABTS were mixed
with water. The compounds were allowed to react for 16 h and then
were diluted by transferring 150 μL into 15 mL ethanol. Trolox
4
.4.2. 1,2-Diacethyl-3-pentadecylbenzene (4)
Yield 82.4%; solid. 1H NMR (CDCl3, 400 MHz, δ, ppm) δ: 7.18 (d,
H, JH-H = 6.9 Hz, CH); 7.12 (t, 1H, JH-H = 7.6 Hz, CH); 7.04 (d, 1H,
(
0–50 μM) was used for the calibration curve.
1
To measure the samples, 30 μL was added to 2000 μL ABTS reagent.
The solution was allowed to stand for 7 min, and the absorbance was
measured at 754 nm.
JH-H = 8.0 Hz, CH); 2.51 (t, 2H, JH-H = 7.6 Hz, CH2); 2.31 (s, 3H,
CH3); 2.27 (s, 3H, CH3); 1.26 (m, 26H, CH2); 0.88 (t, 3H, JH-
H = 6,4 Hz, CH3). 13C NMR (CDCl3, 400 MHz, δ, ppm) δ: 168.7 (s,
C=O); 168.6 (s, C=O); 142.8 (s, C); 140.9 (s, C); 137.1 (s, C); 127.6 (s,
CH); 126.5 (s, CH); 121.2 (s, CH); 32.3 (s, CH2); 30.5–29.7 (m, 12C,
CH2); 23.1 (s, CH2); 21.1 (s, CH3); 20.7 (s, CH3); 14.5 (s, CH3).
4.9. Evaluation of antioxidant activity by FRAP
To prepare the FRAP solution, 25 mL of 300 mM acetate buffer (pH
4
.5. Determination of the inhibitory activity of (Z)-3-(pentadec-10′-enyl)-
3.6) was mixed with 2.5 mL of 20 mM ferric chloride hexahydrate in
distilled water, followed by the addition of 2.5 mL of 2,4,6-tris-(2-
pyridyl)-s-triazine (TPTZ) 10 mM in 40 mM HCl. The samples were
dissolved in methanol, and 1500 μl freshly prepared FRAP solution was
added to each cuvette with 50 μL of the samples dissolved in methanol.
The solution was allowed to stand for 4 min, and absorbance was
measured at 593 nm using a PerkinElmer Lambda 25 UV/Vis spectro-
photometer at room temperature.
catechol (1), 3-pentadecylcatechol (2), (Z)-1,2-diacetyl-3-(pentadec-10′-
enyl)-benzene (3) and 1,2-diacetyl-3-pentadecylbenzene (4) against
soybean 15-lipoxigenase (15-sLOX)
Analysis of 15-sLOX activity (Cayman Chemical Item Nº. 60,712)
−1
−1
was performed at 234 nm (ε = 25.000 M cm ) and was produced
due to the formation of the conjugated diene, which was measured
using a Perkin spectrophotometer-Elmer Lambda 25 UV/Vis. All reac-
tions were carried out with a final volume of 2 mL with constant agi-
tation at room temperature. For this reaction, a 0.1 M HEPES buffer was
prepared at pH 7.4 containing Triton X-100 and 10 μM of the linoleic
acid substrate. IC50 values were determined using the GraphPad Prism
Demo v 8.2.1.
4
.10. Molecular modeling
The crystal structure of 5-hLOX was obtained from the Protein Data
Bank database (PDB) website (http://www.rcsb.org/) in extension
format *.pdb, code 3O8Y (resolution of 2.30 Å) containing an iron atom
in the active site with Fe (III) oxidation state. From this file, the water
molecules present in the macromolecule were eliminated. Using the
AutoDockTools program, hydrogen atoms were added to the protein.
Once these modifications were made, we proceeded to create a file with
extension *. pdbqt, which contained the information generated from
the partial charges and the types of atoms belonging to the macro-
molecule. Ligands were constructed using the GaussView 3.07 program,
and geometric optimization was subsequently carried out using the
Gaussian 09 program. For this optimization, the semi-empirical method
4.6. Determination of the inhibitory activity of (Z)-3- (pentadec-10′-enyl)-
catechol (1), 3-pentadecylcatechol (2), (Z)-1,2-diacetyl-3- (pentadec-10′-
enyl)-benzene (3) and 1,2-diacetyl-3-pentadecylbenzene (1) against human
5-lipoxygenase (5-hLOX) using enzyme kinetics assays
This fluorescence-based assay is performed using microplates. The
enzyme (Cayman Chemical Item No. 60402) was diluted (1:500) in the
assay buffer (HEPES 50 mM, EDTA 2 mM, ATP 10 μM and CaCl
0 μM at pH 7,5) and mixed with 10 μM of H2DCFDA dye. The reaction
mixture was incubated for 15 min in the assay plate. Subsequently,
80 μL of assay buffer was added per well, and 10 μL of inhibitor was
2
1
(
AM1) was initially used followed by the B3LYP method (6-31G*).
Once the structure was optimized and we had determined the par-
2
tial charges derived from electrostatic potential (ESP), molecular cou-
pling analysis was carried out using the Autodock 4 computer program
and the Genetic-Lamarckian algorithm implemented by AutoDock with
a size of grid of 100 points and the box centered on the nonhemic iron
positioned in the catalytic site. Finally, a total of 100 runs were ob-
tained.
added to a final concentration of 10 μM; this reaction mixture was in-
cubated for 30 min. The reaction was started by the addition of a sui-
table concentration of arachidonic acid (0.5 μM), and fluorescence was
read in a multimode detector Synergy™ HT Multi-Mode Microplate
Reader (Biotek) at 480 nm excitation/520 nm emission after a reaction
that had proceeded for 1 h at room temperature for determinate the %I
values. The IC50 values were obtained by the same procedure describe
above but with different inhibitor concentrations (0–10 μM). Finally,
the kinetic assay was performed using different concentrations of sub-
strate (0, 0.5, 1.5 and 3 μM) to determine the inhibitory mechanism and
identify the best inhibitor under the experimental conditions described
previously. All data were collected in duplicate, and the assays were
performed on different days to ensure reproducibility of the methods
used, the GraphPad Prism Demo v 8.2.1 was used for obtaining the
corresponding IC50 (hyperbolic saturation curve) and kinetics para-
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
meters as (K
.7. Evaluation of radical-trapping activity using DPPH
The assay for DPPH uptake capacity was performed in a 96-well
microplate using 20 μL of 2.5 mg/mL compounds in methanol, which
were added to 100 μL of 0.1 mM DPPH in methanol. The plate was
incubated in the dark for 30 min at room temperature. The absorbance
m m i
, V ) by nonlinear regression and K by double-reciprocal.
USA1799 – Vridei 021941 MC-PAP Universidad de Santiago de
Chile. Postgraduate studies financed by CONICYT: CONICYT-PCHA/
Doctorado Nacional/2014–21140089. Powered@NLHPC: This re-
search/thesis was partially supported by the supercomputing infra-
structure of the NLHPC (ECM-02).
4
Appendix A. Supplementary data
(
A) of each reaction mixture was measured at 515 nm and compared
with a blank methanol control using ascorbic acid as the calibration
curve.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112359.
6

A. Muñoz-Ramírez, et al.
Phytochemistry 174 (2020) 112359
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