Fitoterapia 128 (2018) 86–92

Contents lists available at ScienceDirect

Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

Chemical profile and pharmacological effects of the resin and essential oil T
from Bursera slechtendalii: A medicinal “copal tree” of southern Mexico
⁎
Nemesio Villa-Ruanoa, , Yesenia Pacheco-Hernándezb, Elvia Becerra-Martínezc,
José Alejo Zárate-Reyesd, Ramiro Cruz-Duráne
a
Universidad de la Sierra Sur, Guillermo Rojas Mijangos S/N, Ciudad Universitaria, CP 70800, Miahuatlán de Porfirio Díaz Oaxaca, Mexico
b
Centro de Investigación en Biotecnología Aplicada, ex-hacienda San Juan Molino carretera estatal Tecuexcomac-Tepetitla Km 1.5, CP 90700, Tlaxcala, Mexico
c
Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, CP 07738, Ciudad de Mexico, Mexico
d
Centro Universitario de Vinculación y Transferencia de Tecnología, Benemérita Universidad Autónoma de Puebla, CP 72570, Puebla, Mexico
e
Facultad de Ciencias UNAM, Ciudad Universitaria, México DF, CP 04510, Del. Coyoacán, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: This study reports on the volatile profile and pharmacological activities of the essential oil and resin from Bursera
Copal tree slechtendalii, an aromatic medicinal tree commercialized in southern Mexico. The essential oil contained 25
Bursera slechtendalii volatiles including p-cymen-8-ol (25.4–35.7%), n-hexyl hexanoate (6.8–17.6%) and n-octyl hexanoate
Volatiles (7.6–14.8%), whereas the crystallized resin contained 14 volatiles, including n-octyl acetate (29.6–34.2%), li-
Anxiolytic
monene (15.8–18.7%) and n-octyl butyrate (12.6–17-6%). The inhalation of vapors from the resin and essential
Hypocholesterolemic
Insect repellence
oil caused a significant anxiolytic-like effect (p < .01) in ICR mice after 20 min of exposure at a dose of
50–150 mg L−1. The prolonged inhalation of the essential oil (for 40 days, for 15 min, twice a day) produced a
significant decrease (p < .01) in total cholesterol and LDL-c content as well as an increase of HDL-c levels in the
same murine model. Interestingly, levels of hepatic lipid hydroperoxide decreased, whereas the hepatic activity
of superoxide dismutase and glutathione peroxidase was significantly enhanced (P < .05). The detection of p-
cymen-8-ol and n-octyl acetate in the plasma of mice strongly suggests their bioavailability and possible role in
the biological activities described herein. Also, both the resin and essential oil from B. slechtendalii, as well as
their main volatiles, showed a potent repellence activity against Aedes aegypti (p < .01).

1. Introduction role in their relaxant activity, as well as in other health improvements,

In México, the Bursera species are commonly known as “copal trees”
[1,2]. Because of their pleasant smell, the oleoresin and aerial parts of
these plants are mainly used as folk medicines and specially as calming
agents [1,2]. According to recent studies, the resin and aerial parts of
several Bursera species have a promising role in biological and phar-
macological applications [2]. Previous phytochemical screenings per-
formed on Mexican copal trees revealed the presence of terpenes and
lignanes with anti-inflammatory, analgesic, antimicrobial, antitumor
and allelopathic activities [2–4].
In the state of Oaxaca, México, the aromatic resin of Bursera slech-
tendalii Engler (Burseraceae), is often used as a natural anxiolytic.
Intriguingly, the pharmacological activity of the plant has been poorly
investigated [5]. In local markets, the crystallized resin and/or the fresh
leaves of the plant are commercialized and used as a fresh scent in
medicinal baths and also as a natural insect repellent. Considering that
the volatile organic compounds (VOC) of B. slechtendalii may have a
the present study focused in the determination of the VOC from the
resin and essential oil of B. slechtendalii.
2. Results and discussion
2.1. Volatile profile
During the time of our study, the chemical profile of the resin and
essential oil from B. slechtendalii clearly showed significant differences
(p < 0.01) (Table 1; Fig. A.1, Appendix A). The essential oil containing
25 volatiles revealed p-cymen-8-ol (25.4–35.7%) as the most abundant
volatile followed by n-hexyl hexanoate (6.8–17.6%) and n-octyl hex-
anoate (7.6–14.8%). Using these compounds in food industry as flavor
agents is common [6]. On the other hand, the resin containing 14 vo-
latiles showed n-octyl acetate (29.6–34.2%) as the most abundant vo-
latile, followed by limonene (15.8–18.7%) and n-octyl butyrate
(12.6–17-6%). These compounds are also used as natural flavors for

⁎
Corresponding author.
E-mail address: necho82@yahoo.com.mx (N. Villa-Ruano).

https://doi.org/10.1016/j.fitote.2018.05.009
Received 24 March 2018; Received in revised form 3 May 2018; Accepted 12 May 2018
Available online 16 May 2018
0367-326X/ © 2018 Elsevier B.V. All rights reserved.
N. Villa-Ruano et al. Fitoterapia 128 (2018) 86–92

Table 1
Chemical composition of the essential oil (EO) and volatile fraction from the resin (RE) of Bursera slechtendalii, obtained during 2015–2017. Relative abundance is
presented in % ± SD.
Compound IR IR2 EO2015 EO2016 EO2017 RE2015 RE2016 RE2017

a
Nonane 900 900 0.7 ± 0.03 0.6 ± 0.08 1.1 ± 0.05 – – –
α-Thujene 934 933 0.8 ± 0.12 0.8 ± 0.09 3.2 ± 0.43 – – –
a
α-Pinene 936 938 – 0.9 ± 0.07 1.5 ± 0.19 – – –
Sabinene 971 973 0.6 ± 0.06 0 2.2 ± 0.15 – – –
a
β-Pinene 976 979 1.2 ± 0.09 3.6 ± 0.06 0.6 ± 0.04 – – –
β-Myrcene 998 999 1.5 ± 0.08 – 1.8 ± 0.09 – – –
n-Hexyl acetate 1012 1015 2.5 ± 0.07 – 1.7 ± 0.14 – – –
p-Cymene 1024 1025 3.5 ± 0.04 6.6 ± 0.42 0.9 ± 0.06 – 0.7 ± 0.09 1.2 ± 0.18
a
Limonene 1033 1032 2.1 ± 0.05 1.4 ± 0.17 0.5 ± 0.07 18.7 ± 1.9 15.8 ± 1.16 16.8 ± 1.9
a
Eucalyptol 1037 1038 0.7 ± 0.08 1.2 ± 0.16 4.1 ± 0.52 0.5 ± 0.06 – –
Ocimene 1052 1053 – 1.6 ± 0.19 2.3 ± 0.16 0.6 ± 0.08 0.5 ± 0.04
n-Heptyl acetate 1112 1113 – 0.3 ± 0.04 0.6 ± 0.09 1.8 ± 0.14 2.9 ± 0.16 2.7 ± 0.29
a
p-Cymen-8-ol 1180 1179 27.8 ± 1.4 35.7 ± 1.7 25.4 ± 1.6 3.9 ± 0.26 4.3 ± 0.59 3.6 ± 0.43
α-Terpineol 1189 1190 1.1 ± 0.05 0.5 ± 0.09 2.1 ± 0.15 – – –
a
Verbenone 1206 1205 3.3 ± 0.09 – 0.9 ± 0.07 3.8 ± 0.42 2.7 ± 0.32 4.2 ± 0.13
a
n-Octyl acetate 1213 1212 1.5 ± 0.08 – 0.7 ± 0.03 34.2 ± 0.31 29.6 ± 2.17 32.7 ± 1.43
p-Cymene-2,5-dione 1245 1247 6.4 ± 0.09 3.7 ± 0.41 2.9 ± 0.23 0.6 ± 0.03 1.5 ± 0.19 1.9 ± 0.14
a
n-Hexyl hexanoate 1387 1389 17.6 ± 0.08 6.8 ± 0.57 16.6 ± 0.18 5.7 ± 0.42 6.2 ± 0.84 3.8 ± 0.22
a
n-Octyl butyrate 1390 1392 9.1 ± 0.83 7.9 ± 0.32 6.8 ± 0.22 12.6 ± 1.1 17.6 ± 1.9 13.5 ± 1.5
n-Heptyl hexanoate 1462 1465 – 1.4 ± 0.08 2.1 ± 0.08 3.9 ± 0.36 2.9 ± 0.27 2.7 ± 0.26
Elemol 1549 1550 1.1 ± 0.07 2.5 ± 0.06 1.6 ± 0.09 – – –
n-Octyl hexanoate 1573 1575 7.6 ± 0.12 14.8 ± 0.04 10.5 ± 0.6 5.2 ± 0.39 4.5 ± 0.41 5.8 ± 0.59
Spathulenol 1576 1578 3.4 ± 0.14 0.8 ± 0.07 4.2 ± 0.5 4.6 ± 0.37 5.9 ± 0.57 4.7 ± 0.51
Caryophyllene oxide 1581 1583 1.2 ± 0.17 3.7 ± 0.04 – – – –
α-Cadinol 1653 1654 0.6 ± 0.05 1.5 ± 0.06 0.3 ± 0.04 – – –
Monoterpenes 49 56 48.4 28.1 25.5 27.7
Sesquiterpenes 6.3 8.5 6.1 4.6 5.9 4.7
Oxygenated hydrocarbons 39 31.8 40.1 63.4 63.7 61.2
Total 94.3 96.3 94.6 96.1 95.1 93.6

IR1-IR2, Retention index obtained with a HP-5 ms and a ZebronSemivolatiles respectively. Abundances are presented as the average of three different samples plus or
minus standard deviation.
a
Indicates a corroboration by co-injection with authentic standards.

commercial beverages [6]. The chemical profile of the essential oil from
the aerial parts of B. slechtendalii was different to that of the bark es-
sential oils from Bursera grandifolia, B. lancifolia, B. morelensis, B. aptera,
B. glabriifolia, B. vetulina, B. aloexyllon and B. submoniliformis, B. cuneata
and B. simaruba [2,7,8]. Interestingly, the presence of limonene in B.
slechtendalii coincided with the resins of ten Mexican copal trees
[2,7,8]. These results prove that limonene could potentially be a che-
motaxonomical marker for the resins of Bursera sp. Remarkably, arti-
ficial treatments for copal resins (including temperature, age and arti-
sanal process) produce a change in the chemical composition and
volatilization rate [2]. Significant fluctuations were observed in the
main volatiles from the essential oil and resin; however, these com-
pounds were the most abundant in the samples which had been col-
lected for three consecutive years (Fig. A.2, A.3, Appendix A). A higher
variation was observed for minor volatiles (p < .01) (Fig. A.2, A.3,
Appendix A). A previous study on the ethyl acetate extracts of the fresh
resin and leaves of B. slechtendalii showed phellandrene (non-specified
isomer) as the most abundant monoterpene, followed by limonene [3].
Based on our results, the crystallized resin of the plant, which is used as
a botanical anxiolytic, contains a different chemical constitution than
that of fresh resin (with limonene as an exception). Contrary to a pre-
vious chemical screening performed on eight Bursera sp. [8], the con-
tents of diterpenes and sesquiterpenes were negligible in B. slechtendalii
(except for spathulenol in the resin) (Table 1).
2.2. Anxiolytic-like activity of vapors from B. slechtendalii
Resin produced a significant increase (p < .01) in the number of
open-arms entries at 100 and 150 mg L−1 (13.6 ± 0.8 and
15.6 ± 1.1) (Fig. 1A). The effect of vapors from resin had the same
tendency of diazepam, but the activity of the commercial drug was
more potent than that of resin. Only the highest dose (150 mg L−1) of
the essential oil increased this behavioral parameter (Fig. 1A). The
three doses of resin significantly reduced (p < .01) the number of
entries (10.7 ± 1.3–7.6 ± 0.7) into the closed arms which strongly
suggested the anxiolytic-like effect of this component. The highest dose
(150 mg L−1) of the essential oil reduced by ~20% this parameter, in
comparison to the control group (Fig. 1B). Animals subjected to in-
halation doses of 100–150 of resin and 150 mg L−1 of essential oil
showed a significant permanency into the open arms (Fig. 1C). Sur-
prisingly, the three assayed concentrations of the resin and the highest
concentration of the essential oil produced a significant decrease in the
permanency of mice into the closed arms (Fig. 1D). These results sug-
gest that both aromatic components have a potential use as anxiolytic
like agents; however, apparently the resin was more effective than the
essential oil. Other reports on the inhalation of Citrus lemon and rose
essential oils carried out in rodent models, suggest similar anxiolytic-
like effects than those described for the vapors of B. slechtendalii [9,10].
Nevertheless, the assayed doses proved that the essential oil and resin
from B. slechtendalii are more potent than other anxiolytic essential oils
[9,10]. To date, the anxiolytic effect of fresh or crystallized oleo-gum-
resins has been poorly investigated [11]. We believe that ours is the
first scientific study that reveals the anxiolytic-like activity of a Mexican
“copal tree”, which is traditionally used for rituals in southern Mexico.
Interestingly, traces of n-octyl acetate were detected in the plasma of
mice which were exposed for 40 days to the resin of B. slechtendalii
(Fig. 3C–D). This fact strongly suggests the bioavailability of n-octyl
acetate and its potential interaction with diverse physiological targets.
Further experiments could clarify the active mechanisms of the prin-
cipal volatiles from the resin and essential oil from B. slechtendalii.
2.3. Effect of vapors from B. slechtendalii in biochemical parameters of mice
The prolonged inhalation of resin did not cause changes in the

87
N. Villa-Ruano et al.
Fig. 1. Behavior parameters obtained by Elevated-Plus-Maze tests after the inhalation of three different doses of the resin (RE) and essential oil (EO). The components
assayed were those form samples collected and processed in 2015–2016 (RE2015 and EO2016). A, number of entries into open arms. B, number of entries into closed
arms. C, spent time percentage into open arms. D, spent time percentage into closed arms. Diazepam (D) was used as a drug of reference and saline solution (C) was
used as a control of inhalation. The columns and vertical bars represent the means ± SEM of ten mice. * indicates statistically significant differences (p < .01)
compared with the control group.
metabolism of lipids and carbohydrates in the assayed mice (Fig. 2). On
the contrary, exposing the mice only to the essential oil produced a
significant reduction (p < .01) in total cholesterol and LDL-c contents,
as well as an increase in the HDL-c particles (Fig. 2A–C). No significant
reduction was observed in their triglyceride content, glucose levels or
body weight (Fig. 2D–F). Similar results have been observed by only
administering thymol, an isomer of p-cymen-8-ol [12]. It has been
suggested that thymol and related monoterpenes produce an anti-hy-
perlipidemic activity by increasing the HDL-c levels and decreasing
LDL-c levels and low-density lipoprotein cholesterol in the circulation
and membrane stabilization [12]. As expected, an atherogenic diet in-
creased the endogenous levels of hepatic lipid hydroperoxide. Also, a
slight decline (p < .05) was detected from the prolonged inhalation of
the essential oil, which demonstrated its antioxidant properties
(Table 2; Fig. A.4, Appendix A). No significant differences were found
for catalase activity (Table 2; Fig. A.4B, Appendix A); however, a sig-
nificant increase (p < .05) in SOD and GSH-Px activities was observed
(Table 2; Fig. A.4C, A.4D, Appendix A). The analyses of the volatile
fraction of plasma from mice revealed traces of p-cymen-8-ol in the
group treated with essential oil and traces of n-octyl acetate in the
group treated with resin (Fig. 3A–B). The antioxidant and hepatopro-
tective activities of p-cymen-8-ol should be similar to that of its isomer
thymol [13]. According to our results, the bioavailability of the p-
cymen-8-ol in mouse plasma suggests its putative participation in the
scavenging of free radicals and in the enhancement of the endogenous
enzymatic and non-enzymatic antioxidants [12]. Further experiments
are required to endorse this hypothesis.
2.4. Mosquito repellence of vapors from Bursera slechtendalii
The resin and the essential oil had a strong repellence activity
against Aedes aegypti (Fig. 4 A-B). Unlike the essential oil, the resin from
B. slechtendalii produced significant differences in comparison to the
vehicle (p < .01) and to the standard of repellence (p < .05). These
data indicates that resin exerted a stronger activity than DEET (Fig. 4A).
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Fitoterapia 128 (2018) 86–92
The assessment of octyl acetate and p-cymen-8-ol only generated sig-
nificant differences compared to the vehicle (p < .01); however, it was
less effective than DEET (Fig. 4C-D). Surprisingly, hexyl hexanoate and
octyl butyrate produced similar results than those of the resin and es-
sential oil (Fig. 4E–F). However, the synergistic repellence activity of
several volatiles, which were dissolved in the resin and essential oil,
cannot be discarded. The insecticidal and repellence activity of thymol
has been previously reported [14]. Remarkably, the present investiga-
tion is the first evidence that p-cymen-8-ol has a comparable activity to
that of thymol. Despite the allelopathic effect of octyl acetate has been
proved against honey bee [15], our results show for the first time the
repellence effect of octyl acetate, hexyl hexanoate and octyl butyrate on
Aedes aegypti.
3. Conclusions
The volatile fraction of the resin and essential oil of Bursera slech-
tendalii revealed p-cymen-8-ol, n-octyl acetate, n-hexyl hexanoate, li-
monene, n-octyl hexanoate, and n-octyl butyrate as the principal vo-
latiles. These compounds may contribute to the plant's fresh scent. The
vapors of the essential oil and resin produced an anxiolytic-like activity,
while prolonged inhalation generated a hypocholesterolemic effect
which could be associated with an in vivo antioxidant role of p-cymen-8-
ol. The determination of p-cymen-8-ol and n-octyl acetate in the plasma
of the studied mice suggests the possible interaction of this compounds
with biological targets. Finally, the resin, the essential oil and their
main volatiles displayed a potent and prolonged repellence effect
against Aedes aegypti.
4. Materials and methods
4.1. Plant material, resin and essential extraction
For three consecutive years during October (2015–2017), plant
leaves and resin of Bursera slechtendalii Engler (Burseraceae) were
N. Villa-Ruano et al. Fitoterapia 128 (2018) 86–92

Fig. 2. Effect of the prolonged inhalation of vapors from the resin (RE) and essential oil (EO) from Bursera slechtendalii. The components assayed were those form
samples collected and processed in 2015–2016 (RE2015 and EO2016). A, total cholesterol; B, LDL-c content; C, HDL-c content. D, triglyceride content; E glucose
content; F, body weight. The columns and vertical bars represent the means ± SEM of ten mice. * indicates statistically significant differences (p < .01) compared
with the atherogenic group.

Table 2 oil extraction, 1 kg of leaves were cut into small pieces and subjected to
Effect of the prolonged inhalation of vapors from the resin (RE) and essential oil repeating hydrodistillation process (100 g each) using a Clevenger type
(EO) from Bursera slechtendalii on biochemical parameters. The components apparatus (3 h). The essential oil was trapped in n-hexane, then dried
assayed were those form samples collected and processed in 2015–2016 over anhydrous sodium sulfate and finally stored at 4 °C in amber glass
(RE2015 and EO2016). vials until analyzed. The essential oil yield was calculated on a
Group HLHa CAa SODa GSH-Pxa moisture-free basis as 9.87% (w/v). The density of the essential oil was
determined using Drummond microcapillaries (10 μL) as
Standard diet 55.1 ± 2.5 13.7 ± 1.5 58.4 ± 4.2 36.2 ± 3.3
d25 = 0.9211 g cm−3, in accordance with Villa-Ruano et al. [16]. The
Atherogenic diet 82.7 ± 4.1 14.4 ± 1.2 62.7 ± 4.6 38.5 ± 2.8
Atherogenic diet + RE 79.3 ± 4.9 13.2 ± 0.9 60.3 ± 3.7 37.6 ± 2.1 aromatic resin was extracted from braquiblasts and stored in sterile
Atherogenic diet + EO 60.7 ± 1.2⁎ 14.6 ± 1.3 71.4 ± 0.8⁎ 45.9 ± 0.7⁎ glass flasks which were deposited in a glass desiccator until crystal-
lization (for thirty days). Melted resin was obtained at 40 °C for im-
a
HLH, hepatic lipid hydroperoxide levels; CA, hepatic activity of catalase; mediate chromatographic analyses and/or used for in vivo olfactory
SOD, hepatic activity of superoxide dismutase; GSH-Px, activity of glutathione experiments.
peroxidase (mean ± SEM of ten mice). Values are expressed in nmol/mg of
tissue. 4.2. Volatile profiling
⁎
Indicates statistically significant differences (p < .05) compared with both
the atherogenic group (F) and the control group (S).
Analyses of the essential oils ad resin were performed by GC-FID/
GC–MS under the standardized conditions previously reported [16]. For
collected in the district of Miahuatlán de Porfirio Diaz Oaxaca, Mexico
this purpose, the same GC–MS equipment and the capillary columns
(16°13.079′N 096°30.350′W; 1996 masl). A reference voucher 155,370
HP-5 and Zebron ZB-SemiVolatiles were used. The identification of the
was certified by Ramiro Cruz Duran and deposited at the Herbarium of
oil components was achieved according to their mass spectra (70 eV)
the Faculty of Sciences, UNAM, México (FCME-UNAM). Essential oils
and compared with NIST 2.0 Standard Reference database, with the
were obtained from leaf samples and the resins were obtained by
literature [17] and by co-injection of authentic standards (Sigma-Al-
wounding blasquiblasts from five different trees (n = 5). For essential
drich Co). Kovats indices (RI) were obtained by running a standard

89
N. Villa-Ruano et al.
Fig. 3. Detection of main volatiles of B. slechtendalii in the plasma of ICR mice after a prolonged inhalation of essential oil and resin. A, p-cymen-8-ol detected by
GC–MS. B, mass spectrum at 70 eV of p-cymen-8-ol. D, n-octyl acetate detected by GC–MS. E, mass spectrum at 70 eV of n-octyl acetate.
mixture of n-alkanes (C8-C20; Sigma-Aldrich Co.) under similar analy-
tical conditions. The quantification of the components was based on
their GC-FID peak areas. Significant variance of volatiles was validated
by ANOVA-Tukey Test (p < .01) using GraphPad 6.0 software.
4.3. Olfactory experiments in mice
4.3.1. Animals
ICR mice (males weighting 25–32 g) were kept in an air conditioned
room at 25 °C with 50% humidity during 12 h light-dark cycle. Drinking
water was provided ad libitum. Experiments were approved by institu-
tional ethical committee on the basis of the protocol R1627. The tests
were conducted in accordance with the international standards on an-
imal welfare and with the norm NOM-062-ZOO-1999 from Mexico.
4.3.2. Anxiolytic assessment of resin and essential oil vapors in mice
These experiments were carried out in accordance with Viana et al.
[10] with the following modifications. ICR mice (eight week old;
~30 g) were divided into 4 groups of 10 animals (n = 40). The mice of
experimental groups were placed individually into a mass dosing
chamber (DSI™) in which they inhaled, for 20 min, resin previously
melted at 40 °C (25, 50 and 100 mg L−1 of air) and essential oil (25, 50
and 100 mg L−1 of air). After 5 min, the animals were subjected to
behavioral tests. In order to avoid residual smell interference, after each
mouse was tested, the chamber was washed with 10% EtOH. Control
groups were exposed to the inhalation of 1% sodium chloride isotonic
solution and to the intraperitoneal administration of diazepam 1.5 mg/
kg (Compaz®). This drug was administered 30 min before the beha-
vioral tests. Anxiolytic assays were performed using the Elevated Plus-
Maze [10]. An ANOVA coupled to a Dunnett test (p < .01) was used to
determine significant differences between treatments.
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Fitoterapia 128 (2018) 86–92
4.3.3. Prolonged inhalation of resin and essential oil vapors on an
atherogenic diet
Four groups of 10 mice were fed (by oral gavage; 2 mL/kg BW) one
time a day for 40 days using an atherogenic diet [18]. At the same time,
twice a day (in the morning and the evening) mice inhaled the resin
previously melted at 40 °C (150 mg L−1 of air) and the essential oil
(150 mg L−1 of air) for 15 min. Two other control groups were fed ei-
ther an atherogenic diet/water (2 mL/kg BW) or a standard laboratory
diet (LabDiet®)/water (2 mL/kg BW). Both control groups inhaled an
isotonic solution of 1% sodium chloride. At both the beginning and the
end of the experiments, the body weight of mice was measured. At the
end of the treatment period, mice were euthanized with an ether an-
esthesia and their blood was collected in heparinized tubes. Cholesterol
(HDL-c and LDL-c) and triglyceride content were measured using SPI-
NREACT-Cholesterol (HDL-LDL) and SPINREACT-Triglycerides-LQ® kit.
Hepatic samples were removed and extracted with 0.1 M phosphate
buffer, pH 7.4 using a Genie® disruptor. The mixtures were centrifuged
(10,000 g for 15 min) and the supernatant was used to determine the
concentration of hepatic lipid hydroperoxide and activities of anti-
oxidant enzymes catalase, superoxide dismutase (SOD) and glutathione
peroxidase (GSH-Px). These were measured in accordance with An-
drade et al. [19]. The detection of volatiles in plasma was performed by
direct extraction of 25 μL of plasma with 50 μL of n-hexane in triplicate.
Hexane was recovered and concentrated to 10 μL under N2 stream and
then subjected to the GC–MS conditions previously described. An
ANOVA coupled to a Dunnett test (p < .05/p < .01) was used to
determine significant differences between treatments.
4.4. Mosquito repellent assessment of volatiles from the resin and essential
oil
These experiments were done in accordance with Lalthazuali and
Mathew [20], using a laboratory strain of Aedes aegypti. N, N-diethyl-
mmethylbenzamide (DEET) was used a standard of reference.
N. Villa-Ruano et al. Fitoterapia 128 (2018) 86–92

Fig. 4. Repellent activity of vapors from the essential oil and resin from Bursera slechtendalii. The assayed components were those collected and processed during
2017 (EO2017, RE2017). A, effect of 20% resin (20%RE) against vehicle (EtOH) and N, N-diethyl-mmethylbenzamide (20%DEET). B, effect of 20% essential oil (20%
EO) against EtOH and 20%DEET. C, effect of 5% n-octyl acetate (OA5%) against EtOH 20%DEET. D, effect of 5% p-cymen-8-ol (5%CYM) against EtOH and 20%
DEET. E, effect of 5% n-hexyl hexanoate (5%HH) against EtOH and DEET. F, effect of 5% octyl butyrate (5% OB) against EtOH and DEET. Bars represent the standard
deviation of separated experiments carried out in triplicate five times (n = 15). * Indicates statistically significant differences compared to the control group
(vehicle). + Indicates statistically significant differences compared to DEET.

Experiments were performed in separate petri dishes containing a re- References

pellence control (100 μL of 20% DEET), a vehicle control (100 μL EtOH
without repellent) and test solutions (100 μL of 20% essential oil;
100 μL of 20% resin; 100 μL of 5% p-cymen-8-ol; 100 μL of 5% n-octyl
acetate, 100 μL of 5% n-octyl butyrate and n-hexyl hexanoate). Five-
minute landing counts were made every hour for five hours. The per-
centage of mosquitoes that landed was calculated using the formula
described by Lalthazuali1 and Mathew [20]. Experiments were per-
formed in triplicate, five times each. An ANOVA coupled to a Dunnett
test (p < .05/p < .01) was used to determine significant differences
between treatments.
Acknowledgements
We would like to thank M. en C. Javier Luévano Borroel and Dr.
Jorge Ibarra Rendón at CINVESTAV-IPN for the donation of the Aedes
agypti strain. We also thank Max Long for his kind assistance with
proofreading. This work was partially supported by a sabbatical grant
from CONACyT-Mexico (N° Sol. 457483).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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