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Article
Antinociceptive Effect of an Aqueous Extract and Essential Oil
from Baccharis heterophylla
Erika Castillejos-Ramírez 1, Araceli Pérez-Vásquez 1, Rafael Torres-Colín2, Andrés Navarrete 1,
Adolfo Andrade-Cetto 3and Rachel Mata 1, *
Citation: Castillejos-Ramírez, E.;
Pérez-Vásquez, A.; Torres-Colín, R.;
Navarrete, A.; Andrade-Cetto, A.;
Mata, R. Antinociceptive Effect of an
Aqueous Extract and Essential Oil
from Baccharis heterophylla.Plants
2021,10, 116. https://doi.org/
10.3390/plants10010116
Received: 15 December 2020
Accepted: 5 January 2021
Published: 8 January 2021
Publisher’s Note: MDPI stays neu-
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tribution (CC BY) license (https://
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4.0/).
1Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México,
Mexico City 04510, Mexico; erikavivani@gmail.com (E.C.-R.); perezva@unam.mx (A.P.-V.);
anavarrt@unam.mx (A.N.)
2Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico;
rafael.torres@ib.unam.mx
3Laboratorio de Etnofarmacología, Facultad de Ciencias, Universidad Nacional Autónoma de México,
Mexico City 04510, Mexico; aac@ciencias.unam.mx
*Correspondence: rachel@unam.mx; Tel.: +52-55-5622-5289
Abstract:
Infusions and poultices prepared from the aerial parts of Baccharis heterophylla Kunth
(Asteraceae) are widely used in Oaxaca (Mexico) for relieving painful and inflammatory complaints.
Therefore, the antinociceptive potential of an aqueous extract (31.6–316 mg/kg, p.o.) and essential
oil (30–177
µ
g/paw, i.pl.) of the plant was assessed using the formalin test. Both preparations
inhibited the formalin-induced nociception response (100–316 mg/kg and 100–177 µg/paw, respec-
tively) during the test’s second phase. Chemical analysis of the aqueous extract revealed that the
major active components were chlorogenic acid (
1
), 3,4-di-O-(E)-caffeoylquinic acid (
2
), 3,5-di-O-(E)-
caffeoylquinic acid (
3
), 4,5-di-O-(E)-caffeoylquinic acid (
4
), 3,5-di-O-(E)-caffeoylquinic acid methyl
ester (
5
), apigenin (
6
), genkwanin (
7
), acacetin (
8
). Compounds
1
–
5
and
8
are new for B. hetero-
phylla. A high-pressure liquid chromatographic method for quantifying chlorogenic acid (
1
) and
di-caffeoylquinic acids
2
–
4
in the plant was developed and validated. Analyses of the essential
oil and the headspace solid-phase microextraction products, via gas-chromatography-mass spec-
trometry, revealed that the major volatiles were
β
-pinene, myrcene, D-limonene,
β
-caryophyllene,
and α-caryophyllene, which have demonstrated antinociceptive properties.
Keywords:
B. heterophylla; Asteraceae; antinociception; infusion; essential oil; quantification; di-
caffeoylquinic acids; formalin test
1. Introduction
As in many rural regions of the world, the population of Capulálpam de Méndez, a
Zapotec community with well-preserved nature in the state of Oaxaca (Mexico), strongly de-
pends on traditional herbal medicine to meet their primary health care needs [
1
]. This town
has a Traditional Indigenous Medicine Center, which offers healing services to its inhabi-
tants based on regional plants; this practice is firmly anchored in this community’s social
structure and associated with its ancestral culture and history [
2
]. Thus, the medicinal
plants of Capulálpam de Méndez are an essential part of their lives; accordingly, it is
critical to analyze them for the development of evidence-based traditional medicines with
known quality, security, and efficacy.
For instituting knowledge-based herbal traditional medicines, whose use is founded
on the experience, it is necessary to determine their safety and efficacy using preclinical
and clinical assays. When assessing medicinal plants’ efficacy, it is crucial to analyze the
traditional remedies and their constituents (e.g., flavonoids, terpenoids, phenylpropanoids,
alkaloids, or others), considering that usually the efficacy is accomplished with the mixtures
of compounds in these preparations. The mixture of compounds might be acting by syner-
gistic multi-target effects, improving pharmacokinetic parameters or other mechanisms.
Plants 2021,10, 116. https://doi.org/10.3390/plants10010116 https://www.mdpi.com/journal/plants
Plants 2021,10, 116 2 of 13
For quality control of these herbals drugs is important to establish a physicochemical evalua-
tion of crude drug covering aspects, such as selection, macro, and microscopic examination,
for detection of the right material, and search of adulterants; qualitative chemical evalua-
tion for identification of single, or a set of constituents (active or not) using chromatography
or other analytical procedures; and, finally, quantitative chemical evaluation to estimate
the amount of the major classes or constituents, active or not. The processes mentioned in-
volve a wide array of scientific investigations, including physical, chemical, and biological
evaluations employing different analytical methods and tools. Altogether, these studies
can lead to the development of standardized phytomedicines of good quality [3,4].
One of the medicinal species used in Capulálpam de Méndez for painful and inflam-
mation disorders is Baccharis heterophylla Kunth (Asteraceae), a plant widely distributed in
Mexico; the aerial parts of this species are consumed in the form of infusions, decoctions,
or poultices [
5
–
10
]. Baccharis heterophylla is known as “chamizo de barrer”, “curacuata”,
“jarakatua”, and “hierba de la mula”; among others, and it is also employed widely outside
Oaxaca for treating painful complaints. Phytochemical investigations of an exudate from
B. heterophylla recollected in a not specified location of Oaxaca, Mexico, led to the isola-
tion of different types of secondary metabolites, including flavonoids such as apigenin,
genkwanin, and naringenin [
11
], two triterpenes, oleanolic acid, and maniladiol [
11
,
12
],
a few sesquiterpenoids and two dimeric clerodanes [
13
]. Previous pharmacological studies
have shown that the CHCl
3−
MeOH (1:1) extract of B. heterophylla produced a concentration-
dependent inhibition of spontaneous ileum contractions and activated Ca
2+
-dependent
chloride channels in Xenopus laevis oocytes [
6
,
14
]. However, the species’ efficacy to treat
painful complaints, chemical analysis to determine the major compounds of the tradi-
tional preparation, and to establish identity and composition tests remain open questions
Based on the above considerations, the aims of this study were: (i) To investigate the
preclinical efficacy of an aqueous extract (AE), and essential oil (EO) prepared from the
aerial parts of B. heterophylla for treating painful ailments, as well as their chemical composi-
tion. (ii) To develop an analytical method using High-Performance Liquid Chromatography
(HPLC) to quantify the major components of the traditional preparation.
2. Results and Discussion
2.1. Antinociceptive Effect of the Aqueous Extract and Essential Oil
The use of infusions and poultices of B. heterophylla for treating painful complaints led
us to evaluate the antinociceptive activity of an AE and EO obtained from the plant’s aerial
parts using the formalin test. This test is regarded as a satisfactory laboratory model for the
study of nociception as it encompasses inflammatory, neurogenic, and central mechanisms
of nociception. The formalin test is also a useful method for assessing the antinociceptive
drugs and elucidating their action mechanisms [
15
]. The first phase reflects centrally medi-
ated pain with the release of substance P and bradykinin, while the second is due to release
of histamine, serotonin, nitric oxide, bradykinin, and prostaglandins [
15
]. Injection of
2% formalin solution subcutaneously in the hind paw of mice pretreated with vehicle
resulted in intense spontaneous licking of the injected paw with a classic biphasic response
(Figures S1 and S2, Supplementary Materials). However, oral administration of the infusion
(31.6–316 mg/kg) provoked antinociceptive effects in both the first and second phases of
formalin-induced nociception; in the first stage (Figure 1A), the effect was only observed at
the highest dose (316 mg/kg); the highest responses in the second phase was observed at
the doses of 100 and 316 mg/kg, which reduced the licking time significantly. The results
were comparable to the action produced by diclofenac (DIC, 50 mg/kg), employed as
a positive control drug. On the other hand, intraplantar pretreatment of mice with EO
(30–177
µ
g/paw) also decreased the nociceptive response in both phases of the formalin
test (Figure 1B); in the first stage, the highest effect was observed at the concentration of
30
µ
g/paw, but in the second phase the antinociceptive action was more pronounced at the
concentrations of 100 and 177
µ
g/paw, the results were comparable to the effect produced
by positive control (DIC, 100
µ
g/paw). The overall outcomes suggested that the aqueous
Plants 2021,10, 116 3 of 13
extract and essential oil of B. heterophylla exhibited antinociceptive action in both phases of
the formalin test.
Plants 2021, 10, x FOR PEER REVIEW 3 of 13
phases of the formalin test (Figure 1B); in the first stage, the highest effect was observed
at the concentration of 30 µg/paw, but in the second phase the antinociceptive action was
more pronounced at the concentrations of 100 and 177 µg/paw, the results were compa-
rable to the effect produced by positive control (DIC, 100 µg/paw). The overall outcomes
suggested that the aqueous extract and essential oil of B. heterophylla exhibited antino-
ciceptive action in both phases of the formalin test.
VEH DIC 31.6 100 316
0
100
200
300
400
500
600
700
AE (mg/kg)
***
***
*
VEH DIC 30 100 177
0
200
400
600
800
1000
EO ( g/paw)
**
**
*
Figure 1. Antinociceptive effect of an aqueous extract (31.6−316 mg/kg) and an essential oil (30−177 µg/paw) from B. het-
erophylla in the formalin test in mice. (A) AUC from the time course curve of phase 1 and AUC from the time course curve
of phase 2. VEH: vehicle (0.9% saline solution). DIC (50 mg/kg, p.o.). (B) AUC from the time course curve of phase 1 and
AUC from the time course curve of phase 2. VEH: vehicle (0.9% saline solution). DIC: (100 µg/paw, i.pl.). Each measure-
ment represents the mean ± SEM 6 mice per group. Significantly different from VEH group (* p < 0.05, ** p < 0.01, *** p <
0.001) determined by ANOVA followed by Dunnett’s post hoc test.
2.2. Chemical Constituents of the Aqueous Extract
Chemical investigation of the aqueous extract afforded eight known compounds
(Figure 2), identified as chlorogenic acid (1) [16], 3,4-di-O-(E)-caffeoylquinic acid (2) [17],
3,5-di-O-(E)-caffeoylquinic acid (3) [16–18], 4,5-di-O-(E)-caffeoylquinic acid (4) [16–18],
3,5-di-O-(E)-caffeoylquinic acid methyl ester (5) [17], apigenin (6) [19], genkwanin (7) [17],
and acacetin (8) [20]. The structures of the known compounds were identified by compar-
ing their spectroscopic and spectrometric data with those previously reported or by com-
parison with authentic samples (Tables S1 and S2, Supplementary Materials). Compounds
1–5 and 8 are reported for the first time in this species.
(A)
(B)
Figure 1.
Antinociceptive effect of an aqueous extract (31.6
−
316 mg/kg) and an essential oil (30
−
177
µ
g/paw) from
B. heterophylla in the formalin test in mice. (
A
) AUC from the time course curve of phase 1 and AUC from the time course
curve of phase 2. VEH: vehicle (0.9% saline solution). DIC (50 mg/kg, p.o.). (
B
) AUC from the time course curve of
phase 1 and AUC from the time course curve of phase 2. VEH: vehicle (0.9% saline solution). DIC: (100
µ
g/paw, i.pl.).
Each measurement represents the mean
±
SEM 6 mice per group. Significantly different from VEH group (* p< 0.05,
** p< 0.01, *** p< 0.001) determined by ANOVA followed by Dunnett’s post hoc test.
2.2. Chemical Constituents of the Aqueous Extract
Chemical investigation of the aqueous extract afforded eight known compounds
(Figure 2), identified as chlorogenic acid (
1
) [
16
], 3,4-di-O-(E)-caffeoylquinic acid (
2
) [
17
],
3,5-di-O-(E)-caffeoylquinic acid (
3
) [
16
–
18
], 4,5-di-O-(E)-caffeoylquinic acid (
4
) [
16
–
18
],
3,5-di-O-(E)-caffeoylquinic acid methyl ester (
5
) [
17
], apigenin (
6
) [
19
], genkwanin (
7
) [
17
],
and acacetin (
8
) [
20
]. The structures of the known compounds were identified by comparing
their spectroscopic and spectrometric data with those previously reported or by comparison
with authentic samples (Tables S1 and S2, Supplementary Materials). Compounds
1
–
5
and
8are reported for the first time in this species.
Plants 2021,10, 116 4 of 13
Plants 2021, 10, x FOR PEER REVIEW 4 of 13
Figure 2. Compounds isolated from B. heterophylla’s aqueous extract.
As with other plant’s traditional preparations [21–29], the antinociceptive effects of
AE could be related to the high content of chlorogenic acid (1), di-O-(E)-caffeoylquinic
acids (2–4), apigenin (6) and acacetin (8). Thus, in vivo studies demonstrated that com-
pounds 3,5-di-O-(E)-caffeoylquinic acid (3) and 4,5-di-O-(E)-caffeoylquinic acid (4)
showed antinociceptive action in the acetic acid-induced writhing model and the hot plate
assay in mouse [21,22]. Compounds 1, 3, and 4 significantly inhibited carrageenan-in-
duced rat paw edema [23,24]. In vitro, compound 4 inhibited hypoxia-induced cyclooxy-
genase-2 (COX- 2), expression and cell migration via a TRPV1-mediated pathway [25];
pretreatment of RAW264.7 macrophage cells with a mixture of di-caffeoyl quinic acids or
pure 3 suppressed the production of NO, PGE2, and pro-inflammatory cytokines (TNF-
α, IL-1β, and IL-6) by inhibiting the NF-κB and MAPKs pathways ([26−28] inter alia). On
the other hand, flavonoids 6 and 8 showed antinociceptive effects in different models [29].
2.3. Volatile Composition Analyses
The volatile components profile of B. heterophylla was gathered by gas
chromatography-mass spectrometry (GC-MS) analyses of EO obtained by
hydrodistillation and head space-solid phase microextraction (HS–SPME)-adsorbed
compounds. The HS-SPME is a more efficient, quicker, and free-solvent method for ana-
lyzing volatile constituents from plants and other matrices [30].
The results of the study of EO are summarized in Table 1 and Figure 3. The EO of B.
heterophylla is characterized by a high concentration of monoterpenes, such as β-pinene
(28.86%), myrcene (29.57%), and D-limonene (36.24%). These results agree with the
chemical composition of essential oils reported for other species of the genus Baccharis
[31].
For the HS-SPME analysis, four fibers were employed, carboxen/polydimethylsilox-
ane (CAR/PDMS), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS),
polydimethylsiloxane/divinylbenzene (PDMS/DVB), and polydimethylsiloxane (PDMS).
As summarized in Table 1, the analysis with these fibers allowed the identification of 13,
13, 14, and 14 compounds, respectively; their relative percentages and retention indexes
are also indicated in Table 1. β-Pinene, D-limonene, and myrcene were the major compo-
nents detected with DVB/CAR/PDMS and CAR/PDMS stationary phases; this composi-
tion is consistent with the main compounds detected in EO. On the other hand, cedrene
and β-caryophyllene were the most abundant compounds detected with PDMS and
PDMS/DVB-coat fibers.
Figure 2. Compounds isolated from B. heterophylla’s aqueous extract.
As with other plant’s traditional preparations [
21
–
29
], the antinociceptive effects of
AE could be related to the high content of chlorogenic acid (
1
), di-O-(E)-caffeoylquinic
acids (
2
–
4
), apigenin (
6
) and acacetin (
8
). Thus,
in vivo
studies demonstrated that com-
pounds 3,5-di-O-(E)-caffeoylquinic acid (
3
) and 4,5-di-O-(E)-caffeoylquinic acid (
4
) showed
antinociceptive action in the acetic acid-induced writhing model and the hot plate assay in
mouse [
21
,
22
]. Compounds
1
,
3
, and
4
significantly inhibited carrageenan-induced rat paw
edema [
23
,
24
].
In vitro
, compound
4
inhibited hypoxia-induced cyclooxygenase-2 (COX-
2), expression and cell migration via a TRPV1-mediated pathway [
25
]; pretreatment of
RAW264.7 macrophage cells with a mixture of di-caffeoyl quinic acids or pure
3
suppressed
the production of NO, PGE2, and pro-inflammatory cytokines (TNF-
α
, IL-1
β
, and IL-6)
by inhibiting the NF-
κ
B and MAPKs pathways ([
26
–
28
] inter alia). On the other hand,
flavonoids 6and 8showed antinociceptive effects in different models [29].
2.3. Volatile Composition Analyses
The volatile components profile of B. heterophylla was gathered by gas chromatography-
mass spectrometry (GC-MS) analyses of EO obtained by hydrodistillation and head space-
solid phase microextraction (HS–SPME)-adsorbed compounds. The HS-SPME is a more ef-
ficient, quicker, and free-solvent method for analyzing volatile constituents from plants
and other matrices [30].
The results of the study of EO are summarized in Table 1and Figure 3. The EO
of B. heterophylla is characterized by a high concentration of monoterpenes, such as
β
-
pinene (28.86%), myrcene (29.57%), and D-limonene (36.24%). These results agree with the
chemical composition of essential oils reported for other species of the genus Baccharis [
31
].
For the HS-SPME analysis, four fibers were employed, carboxen/polydimethylsiloxane
(CAR/PDMS), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), poly-
dimethylsiloxane/divinylbenzene (PDMS/DVB), and polydimethylsiloxane (PDMS). As sum-
marized in Table 1, the analysis with these fibers allowed the identification of 13, 13, 14, and
14 compounds, respectively; their relative percentages and retention indexes are also indi-
cated in Table 1.
β
-Pinene, D-limonene, and myrcene were the major components detected
with DVB/CAR/PDMS and CAR/PDMS stationary phases; this composition is consistent
with the main compounds detected in EO. On the other hand, cedrene and
β
-caryophyllene
were the most abundant compounds detected with PDMS and PDMS/DVB-coat fibers.
Plants 2021,10, 116 5 of 13
Table 1. Volatile constituents from B. heterophylla identified by GC-MS obtained by HS-SPME using different fibers and by hydrodistillation.
N◦Compound IRPercent of Each Component
EO ** PDMS * PDMS/DVB * DVB/CAR/PDMS * CAR/PDMS *
9α-Pinene 922 5.34 - - - -
10 β-Pinene 969 28.86 4.29 4.85 10.6 1.97
11 Myrcene 982 29.57 3.43 9.4 27.19 10.53
12 o-Cymene 1016 - - - - 26.71
13 D-Limonene 1019 36.24 4.07 8.82 23.02 26.71
14 p-Cymenene 1081 - - - - 3.18
15 δ-Elemene 1327 2.45 1.34 - -
16 Cedrene 1413 - 19.99 15.62 - -
17 β-Caryophyllene 1414 - 19.99 15.62 9.25 4.23
18 Isogermacrene D 1422 - 8.51 5.12 1.89 -
19 Aromadendrene 1435 - 3.64 4.01 - 1.92
20 α-Caryophyllene 1447 - 7.37 6.95 1.72 5.03
21 Germacrene D 1454 - 1.14 1.15 - -
22 γ-Elemene 1470 - 5.6 4.45 8.59 2.7
23 α-Selinene 1479 - 1.04 - - -
24 β-Cadinene 1486 - - - - 4.46
25 β-Amorphene 1488 - - - 4.77 4.32
26 β-Himachalene 1493 - 9.72 8.46 1.27 -
27 δ-Amorphene 1510 - - 3.53 4.3 -
28 Calamenene 1511 - - - 4.3 3.92
29 α-Cadinene 1525 - - - 2.45 1.6
30 α-Calacorene 1531 - 0.39 0.4 0.64 -
Total (%) 100 91.63 89.72 99.99 97.28
IR: Index retention on a DB-5 column with reference to n-alkanes. * Fibers used for HS-SPME. ** Obtained by hydrodistillation.
Plants 2021,10, 116 6 of 13
Plants 2021, 10, x FOR PEER REVIEW 5 of 13
Figure 3. Total ion current chromatogram of the components of the essential oil from B. hetero-
phylla extracted using hydrodistillation. Peaks are: α-pinene (9), β-pinene (10), myrcene (11) and D-
limonene (12).
Altogether, these results revealed that the mixed polarity DVB/CAR/PDMS fiber was
the most suitable to establish the volatile composition of the plant’s aerial parts, which is
characterized by a high number of hydrocarbons. The total ionic chromatograms of the
volatile compounds obtained using this technique are shown in the supplementary mate-
rials (Figures S3 and S10).
The major components of the antinociceptive essential oil of B. heterophylla are pre-
sent in several essences, which showed antinociceptive properties [32–36]. β-Pinene (0.3
mg/kg) exhibited antinociceptive action in the hot-place and tail-flick models in rodents
[32]. α-Pinene decreased the LPS-induced production of interleukin-6 (IL-6), tumor necro-
sis factor-α (TNF-α) and inhibited the expression of inducible nitric oxide synthase (iNOS)
and cyclooxygenase-2 (COX-2) [33]. At a dose of 10 mg/kg, myrcene was effective in the
formalin and hot-plate tests [34]. Besides, myrcene and limonene inhibited IL-1β-induced
nitric oxide production in human chondrocytes [35]. Finally, limonene exerted anti-in-
flammatory and antinociceptive effects in vivo (particularly in chemical models of noci-
ception in mice) and in vitro assays mainly by modulating the action of cytokines and
participating in pathways that are closely linked to the inflammatory response [36].
2.4. Method Validation
In the present study, chlorogenic and di-caffeoylquinic acids were selected as mark-
ers for the quantitative analysis of B. heterophylla. The optimal chromatographic separation
was achieved with a Waters HPLC equipped with an XBridge BEH Shield RP18 column
and a mixture of water containing 0.1% formic acid and acetonitrile as mobile phase,
which was chosen after several trials; the PDA detection wavelength (λ) was set at 327
nm; under these optimized conditions, an effective resolution was achieved for com-
pounds 1–4. Compounds 1–4 were identified by their retention time and coelution with
AE; a representative chromatogram of the AE of B. heterophylla (BH-1) is shown in Figure
4. The developed method was validated using compounds 1, 3 and 4 according to the
International Conference on Harmonization guideline (ICH) [37], and was successfully
applied to determine the content of caffeoylquinic acids in AE prepared from different
batches of the plant. The overall results are summarized in Tables 2 and 3.
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
900.0
1000.0
1100.0
1200.0
250.0 350.0 450.0 550.0 650.0
Time (s)
9
10
11 12
Figure 3.
Total ion current chromatogram of the components of the essential oil from B. heterophylla
extracted using hydrodistillation. Peaks are:
α
-pinene (
9
),
β
-pinene (
10
), myrcene (
11
) and D-
limonene (12).
Altogether, these results revealed that the mixed polarity DVB/CAR/PDMS fiber
was the most suitable to establish the volatile composition of the plant’s aerial parts,
which is characterized by a high number of hydrocarbons. The total ionic chromatograms
of the volatile compounds obtained using this technique are shown in the supplementary
materials (Figures S3 and S10).
The major components of the antinociceptive essential oil of B. heterophylla are present
in several essences, which showed antinociceptive properties [
32
–
36
].
β
-Pinene (0.3 mg/kg)
exhibited antinociceptive action in the hot-place and tail-flick models in rodents [
32
].
α
-Pinene decreased the LPS-induced production of interleukin-6 (IL-6), tumor necrosis
factor-
α
(TNF-
α
) and inhibited the expression of inducible nitric oxide synthase (iNOS)
and cyclooxygenase-2 (COX-2) [
33
]. At a dose of 10 mg/kg, myrcene was effective in
the formalin and hot-plate tests [
34
]. Besides, myrcene and limonene inhibited IL-1
β
-
induced nitric oxide production in human chondrocytes [
35
]. Finally, limonene exerted
anti-inflammatory and antinociceptive effects
in vivo
(particularly in chemical models of
nociception in mice) and
in vitro
assays mainly by modulating the action of cytokines and
participating in pathways that are closely linked to the inflammatory response [36].
2.4. Method Validation
In the present study, chlorogenic and di-caffeoylquinic acids were selected as markers
for the quantitative analysis of B. heterophylla. The optimal chromatographic separation was
achieved with a Waters HPLC equipped with an XBridge BEH Shield RP18 column and a
mixture of water containing 0.1% formic acid and acetonitrile as mobile phase, which was
chosen after several trials; the PDA detection wavelength (
λ
) was set at 327 nm; under these
optimized conditions, an effective resolution was achieved for compounds
1
–
4
. Com-
pounds
1
–
4
were identified by their retention time and coelution with AE; a representative
chromatogram of the AE of B. heterophylla (BH-1) is shown in
Figure 4
. The developed
method was validated using compounds
1
,
3
and
4
according to the International Confer-
ence on Harmonization guideline (ICH) [
37
], and was successfully applied to determine
the content of caffeoylquinic acids in AE prepared from different batches of the plant.
The overall results are summarized in Tables 2and 3.
Plants 2021,10, 116 7 of 13
Plants 2021, 10, x FOR PEER REVIEW 7 of 13
Figure 4. HPLC-PDA chromatogram of B. heterophylla aqueous extract under optimized condi-
tions; detection wavelength at 327 nm. Peak identification: 1: RT 6 min; 2: RT 14.6 min; 3: RT 15.6
min; 4: RT 16.2 min; 5: RT 17.2 min; 6: RT 23.6 min.
The method’s selectivity was determined by comparing the chromatographic profile
of AE with the data obtained for the standards, considering the retention time and UV
spectra (Figures S4–S6, Supplementary Materials). The linearity was tested by analyzing
a series of different concentration ranges: 5 to 150 µg/mL for 1, 20 to 200 µg/mL for 3, and
13.4 to 133.4 µg/mL for 4. In all cases, the value of the determination coefficient (R2) was
greater than 0.99. The limits of detection (LOD) and quantification (LOQ) values were 0.5
µg/mL and 1.5 µg/mL for compound 1, 2.2 µg/mL and 6.6 for compound 3 and 1.4 µg/mL
and 4.1 µg/mL for compound 4. The intraday and interday precision RSDs were no more
than 2% while the repeatability variation was no more than 2% (Table 2). Percentage re-
coveries of the standards are also indicated in Table 2; in each case, a good accuracy was
obtained in the range from 99.15% to 100.83% (RSD ≤ 2.0%). Altogether the described pre-
vious data revealed that the method was linear, precise and accurate in the range of con-
centrations evaluated.
The validated method was applied successfully to quantify chlorogenic acid deriva-
tives in four different batches (BH-1, BH-2, BH-3, and BH-4) of the crude drug. The results
are presented in Table 3, which shows the content of 1–4 was similar in all batches, where
3,5-di-O-(E)-caffeoylquinic acid was the major component.
Table 3. Contents (mg/g) compounds in four batches of the B. heterophylla (n = 3).
Compound RT (min) Content (mg/g)
BH-1 BH-2 BH-3 BH-4
1 6 37.9 ± 3.4 39.7 ± 1.7 29.3 ± 1.8 33.9 ± 2.1
2 a 14.6 52.4 ± 1.4 61.5 ± 4.5 58.8 ± 3.4 53.1 ± 1.4
3 15.6 104.7 ± 3.4 107 ± 12.3 79.8 ± 8.4 99.6 ± 4.4
4 16.2 42.1 ± 1.2 44.5 ± 4.3 40.6 ± 4.3 29.3 ± 0.3
a Quantified as 4.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
024681012141618202224262830
AU
Time (min)
1
2
3
4
56
Figure 4.
HPLC-PDA chromatogram of B. heterophylla aqueous extract under optimized conditions;
detection wavelength at 327 nm. Peak identification:
1
:R
T
6 min;
2
:R
T
14.6 min;
3
:R
T
15.6 min;
4:RT16.2 min; 5:RT17.2 min; 6:RT23.6 min.
Table 2. Parameters of validation of method for determination of constituents in B. heterophylla.
N◦Linear Range
(µg/mL) Calibration Equation R2LOQ
(µg/mL)
LOD
(µg/mL)
Precision Recovery
(% Mean)
Intraday
(% RSD)
Interday
(% RSD)
15–150 y = 84,372x + 67,400
0.999
1.5 0.5 0.91 1.32 100.83
320–200 y = 93,446x −216,607
0.997
6.6 2.2 1.22 1.8 99.15
413.4–133.4 y = 72,602x −156,669
0.996
4.1 1.4 1.96 2.0 100.4
Table 3. Contents (mg/g) compounds in four batches of the B. heterophylla (n= 3).
Compound RT(min) Content (mg/g)
BH-1 BH-2 BH-3 BH-4
16 37.9 ±3.4 39.7 ±1.7 29.3 ±1.8 33.9 ±2.1
2a14.6 52.4 ±1.4 61.5 ±4.5 58.8 ±3.4 53.1 ±1.4
315.6 104.7 ±3.4 107 ±12.3 79.8 ±8.4 99.6 ±4.4
416.2 42.1 ±1.2 44.5 ±4.3 40.6 ±4.3 29.3 ±0.3
aQuantified as 4.
The method’s selectivity was determined by comparing the chromatographic profile
of AE with the data obtained for the standards, considering the retention time and UV
spectra (Figures S4–S6, Supplementary Materials). The linearity was tested by analyzing
a series of different concentration ranges: 5 to 150
µ
g/mL for
1
, 20 to 200
µ
g/mL for
3
,
and 13.4 to 133.4
µ
g/mL for
4
. In all cases, the value of the determination coefficient (R
2
)
was greater than 0.99. The limits of detection (LOD) and quantification (LOQ) values
were 0.5
µ
g/mL and 1.5
µ
g/mL for compound
1
, 2.2
µ
g/mL and 6.6 for compound
3
and
1.4
µ
g/mL and 4.1
µ
g/mL for compound
4
. The intraday and interday precision RSDs
were no more than 2% while the repeatability variation was no more than 2% (Table 2).
Percentage recoveries of the standards are also indicated in Table 2; in each case, a good
accuracy was obtained in the range from 99.15% to 100.83% (RSD
≤
2.0%). Altogether the
described previous data revealed that the method was linear, precise and accurate in the
range of concentrations evaluated.
Plants 2021,10, 116 8 of 13
The validated method was applied successfully to quantify chlorogenic acid deriva-
tives in four different batches (BH-1, BH-2, BH-3, and BH-4) of the crude drug. The re-
sults are presented in Table 3, which shows the content of
1
–
4
was similar in all batches,
where 3,5-di-O-(E)-caffeoylquinic acid was the major component.
3. Materials and Methods
3.1. General Experimental Procedures
Melting points were determined on a Fisher-Johns apparatus (Thermo Scientific,
Vernon Hills, IL, USA) and are uncorrected. NMR spectra were recorded in a Unity Plus
400 spectrometer (Varian, Palo Alto, CA, USA), at either 400 MHz (
1
H) or 100 (
13
C) MHz,
in DMSO-d
6
and MeOH-d
4
. Data processing was carried out with the software MestReNova
version 12.0.0. Mass spectra of the isolates were obtained on an Acquity UHPLC-H Acquity
UHPLC-H
®
Class system (Waters, Milford, MA, USA) equipped with a quaternary pump,
sample manager, column oven and photodiode array detector (PDA) interfaced with an
SQD2 single mass spectrometer detector with an electrospray ion source. IR spectra were
recorded using a Spectrum RXI FTIR (Perkin-Elmer, Waltham, MA, USA). Open column
chromatography was carried out on Sephadex LH-20 (GE Healthcare, Urbandale, IA, USA)
and silica gel 60, 70-230 mesh (Merck, Darmstadt, Germany).
3.2. Reagents
HPLC grade acetonitrile and water, and analytical reagent (AR) grade solvents used for
the chromatographic processes were purchased from J.T. Baker (Avantor,
Radnor, PA, USA
).
Chlorogenic acid (1), diclofenac, and Tween 80 were purchased from Sigma-Aldrich
(
St. Louis, MO, USA
). Standards (
3
and
4
) used in the method validation were isolated
from B. heterophylla (purities ≥97%).
3.3. Plant Material
Baccharis heterophylla was collected in Capulálpam de Méndez, Oaxaca, México on
April 2019 (BH-1). The plant material was identified by Rafael Torres Collin and a voucher
specimen (1512112) was deposited at the National Herbarium (MEXU), UNAM, Mex-
ico City. In addition, batches from February, July, and October (BH-2, BH-3 and BH-4
respectively) were collected in the same place and the same year, to analyze their content.
3.4. Preparation of the Aqueous Extract and Essential Oil
Air-dried aerial parts (leaves and stem) of B. heterophylla (5 g) were extracted with
250 mL of boiling water for 30 min. The resulting infusion was filtrated and concentrated
in vacuo to yield 1.28 g of dry aqueous extract (EA). On the order hand, the infusion was
exhaustively partitioned with EtOAc (250 mL
×
3); the combined organic fractions were
dried over anhydrous sodium sulfate and concentrated in vacuo to yield 0.116 g of dry
ethyl acetate fraction. This process was repeated as much as needed to obtain 10 g of
this fraction.
Essential oil (EO) was prepared by hydrodistillation (3 h), with 200 g of fresh aerial
parts and 1.3 L of distilled water, using a Clevenger-type instrument. The hydrodistilled
mixture was extracted with dichloromethane (250 mL
×
3); the combined organic phases
were dried over anhydrous sodium sulfate and concentrated in vacuo. This procedure was
performed in triplicate and the resulting oils (320 mg) were stored at
−
4
◦
C until chemical
analysis and pharmacological experiments.
3.5. Pharmacological Study
3.5.1. Animals
Male CD1 mice weighting between 30–38 g were obtained from the Bioterium of
the School of Sciences, UNAM. Animals were kept on a 12 h light/dark cycle under
controlled temperature (22
±
1
◦
C) and given a standard pellet diet ad libitum until the
beginning of each experiment. Animal experimental protocols followed recommenda-
Plants 2021,10, 116 9 of 13
tions of the Mexican Official Norm Animal Care and Handling (NOM-062-ZOO-1999)
and were in conformity with international ethical guidelines form care and use of lab-
oratory animals. The experimental protocols were approved by the Institutional Com-
mittee for Care and Use of Laboratory Animals (CICUAL-FQ) of Facultad de Química,
UNAM (FQ/CICUAL/391/19).
3.5.2. Antinociceptive Effect
The antinociceptive effect of AE and EO was assessed using the formalin test [
38
].
All the samples were diluted in the vehicle and animals were divided into groups (n= 6).
Mice were treated with AE (31.6–316 mg/kg, p.o.), EO (30–170
µ
g/paw, i.pl.), diclofenac
(positive control, 50 mg/kg, p.o. and 100
µ
g/paw, i.pl.) or with vehicle (0.5% Tween 80 in
0.9% saline solution). After 30 min, each animal was received 30
µ
L of diluted 2% formalin
in into the mice dorsal surface of the right hind paw. The biphasic response (licking of
the injected paw) induced by the formalin solution was quantified every 5 min during a
30 min period [
13
]. The doses were logarithmic, and were selected on the basis of previous
experimental design for traditional preparations [38].
3.5.3. Statistical Analyses
Antinociceptive results are expressed as the mean
±
S.E.M. of the analysis of area
under the curve (AUC, time of licking against time, sec
×
min) of mice (n= 6), for phases 1
and 2, and both, or as the mean
±
S.E.M. of licking time (sec) of mice (n= 6) in time courses
(Supplementary Materials). Statistical differences were evaluated using either one way
ANOVA followed by Dunnett’s in the GraphPad Prism software (version 7; GraphPad Inc.,
La Jolla, CA, USA).
3.6. Isolation of Compounds
The ethyl acetate fraction (9.5 g) was subjected to column chromatography on Sephadex
LH-20, eluting with methanol; 28 (16 mL each) fractions were obtained (F
1
–F
28
). Sec-
ondary fraction F
20
was dissolved in MeOH; from this solution precipitated 85 mg of
apigenin (
6
). Column chromatography on Sephadex LH-20 of F
16
(1.20 g), eluting with
acetone
−
MeOH (9:1), rendered 24 fractions (8 mL each): F
16-1
–F
16-24
. Fractions F
16-5
, F
16-10
,
and F
16-13
yielded 14 mg of
3
, 8 mg of
5
and 5 mg of
4
, respectively. Fraction F
17
(50 mg)
was subjected to column chromatography on silica gel (50 g), eluting with a gradient of
hexane
−
EtOAc (9:1
→
0:10) and EtOAc
−
MeOH (10:0
→
0:10), 12 fractions of 300 mL each
were obtained. From F
17-5
, eluted with hexane
−
EtOAc (7:3), 9 mg of
7
were obtained.
From F
17-6
, eluted with hexane
−
EtOAc (6:4), crystalized 8 mg of
8
. Compounds
1
and
2
were identified by comparison with authentic standards (TLC, HPLC, NMR).
3.7. GC-MS Analyses
All analyses by GC-MS were carried out in an Agilent 6890 N (Agilent Technol-
ogy, Santa Clara, CA, USA) series gas chromatograph equipped with a LECO Pegasus
4D time-of-flight mass spectrometer detector (IET International Equipment Trading Ltd.,
Mundelein, IL, USA
). A capillary column DB-5 was used [(5%-phenyl)-methylpolysiloxane,
20 m ×0.18 mm I.D.
; 0.18
µ
m film thickness]. The oven temperature was set at 40
◦
C for
three minutes, then at 40 to 300
◦
C at 20
◦
C/min and held isothermally at 300
◦
C for 15 min.
Helium was used as the carrier gas at a flow rate of 1 mL/min. Compounds were identified
using index retention method (I
R
), by co-injection of the sample with a solution containing
the homologous series of n-alkanes (C
8−
C
27
), and by comparison of their MS fragmenta-
tion patterns with those of compounds contained in the spectral database of the National
Institute of Standards and Technology (NIST,
Gaithersburg, MD, USA
). All determinations
were performed in triplicate.
Plants 2021,10, 116 10 of 13
3.8. Headspace Solid-Phase Microextraction
Volatile compounds of B. heterophylla were extracted using the HS-SPME technique; the
extraction procedure was conducted as follows: 50 mg of aerial parts, 2 g of sodium chloride,
and 15 mL of water (HPLC grade) were mixed in a hermetically sealed vial. Each fiber
(CAR/PDMS, DVB/CAR/PDMS, PDMS/DVB and PDMS) was introduced into the vial
and exposed to the headspace of the sample for 15 min, at room temperature, keeping the
sample magnetically stirred. After sampling, the SPME fibers were directly inserted into
the GC injector port and the fibers thermally desorbed. A desorption time of 2 min at
250
◦
C was used. Before GC–MS analysis, the fibers were conditioned in the injector of the
GC system, according to the instructions provided by the manufacturer. All samples were
analyzed in triplicate and the relative proportions of individual components adsorbed
to the fibers under these conditions was calculated based on the total ion chromatogram
(TIC) peak areas as a percentage of the sum of all peak areas. The GC-MS conditions were
described in Section 3.7. The SPME fibers used in this study were purchased from Supelco
Inc. (Bellefonte, PA, USA).
3.9. HPLC Analyses
All experiments were performed on a Waters HPLC system equipped with a quater-
nary pump (model 600), photodiode array detector (PDA, model 996), manual injector
and an XBridgeTM BEH Shield RP18 column, (130 Å, 5
µ
m, 4.6 mm
×
250 mm, Waters)
at a flow rate of 0.8 mL/min. The mobile phase consisted of (A) acetonitrile and (B) wa-
ter (0.1% formic acid) the following gradient elution program was used: 20–40% A for
0–10 min
, 40–100% A for 10–23 min, 100% A for 23–24 min, and 100–20% A for
24–28 min
,
20% A
for 28–30 min, the injection volume was 20
µ
L. The UV detector was set at a moni-
toring wavelength of 327 nm. System control, data collection, and data processing were
accomplished using Waters Empower 2 chromatography software.
3.10. Method Validation
The HPLC method was validated according to the International Conference on Har-
monization Guidelines (ICH, 2005) and included a determination of selectivity, linearity,
precision, accuracy, LOD, and LOQ. The linearity of the system was performed through
the calibration curves of the standards (
1
,
3
, and
4
); compounds were accurately weighed
(5 mg) and dissolved in 5 mL acetonitrile-water (5 mL, 1:5) to prepare stock solutions at a
final concentration of 1 mg/mL. The solutions of the calibration curve were obtained by
diluting the stock solution in water, ranging from 5
−
150
µ
g/mL for
1
, 20
−
200
µ
g/mL for
3
and 13.3
−
133.4
µ
g/mL for
4
. The linearity was assessed estimating the slope, y-intercept,
and the determination coefficient (R
2
) using the least-squares analysis. LOD and LOQ were
determined based on the standard deviation (
σ
) of the response and the slope (S) from
calibration curve constructed with a series of appropriate concentrations for determination
of the limits, using the following equations:
LOD =3.3 σ
S(1)
LOQ =10 σ
S(2)
The precision was evaluated using repeatability (intraday) and intermediate preci-
sion (interday). Intraday and interday variations were established using six independent
replicates of the standard reference of each compound on one and two different days,
to determine intraday and interday precision, respectively. In order to study the accu-
racy of the method, recovery experiments were performed. The samples were spiked with
known amounts of the standards, at different concentrations levels (low, medium and high):
Plants 2021,10, 116 11 of 13
1
(5, 50 and 150
µ
g/mL),
3
(20, 100 and 200
µ
g/mL) and
4
(13.3, 66.8 and 133.4
µ
g/mL).
The average recoveries were calculated according to the following formula:
Recovery (%) = amount f ound −original amount
amount spiked ×100 (3)
The relative standard deviation (RSD) was calculated for each determination as a
measure of precision and repeatability.
4. Conclusions
In summary, the aerial parts of B. heterophylla exhibited antinociceptive activity when
tested by the formalin assay. The main classes of compounds characterized in the traditional
preparation were chlorogenic acid derivatives and flavonoids with known antinocicep-
tive properties. In the case of the essential oil, the activity could be attributed to its
high content of antinociceptive monoterpenes. In any case, the pharmacological effects
might be achieved via pharmacology synergism and/or polypharmacology. A precise,
reliable, and accurate HPLC method for quantifying chlorogenic acid derivatives’ content
in the infusion of the plant was developed and validated. This methodology will be use-
ful for preparing standardized infusions or other formulations made up of this species.
This method will also be proposed as composition test for the monograph of B. heterophylla
to be included in the new edition of the Mexican Herbal Pharmacopoeia. Altogether, our
results tend to support the medicinal use of B. heterophylla to treat painful complaints in
Mexican folk medicine and contribute to the rational use of this valuable medicinal plant.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2223-7
747/10/1/116/s1,
Figure S1
: Temporal course of the antinociceptive behavior (the licking time
against time) and AUC from the time course curve for the Aqueous Extract (AE, 31.6–316mg/kg).
Each measurement represented as mean
±
SEM of n= 6. Significantly different from VEH group
(
** p< 0.01
,
*** p< 0.001
) determined by ANOVA followed by Dunnett’s post hoc test,
Figure S2
:
Temporal course of the antinociceptive behavior (the licking time against time) and AUC from the
time course curve for the Essential Oil (EO, 30–177
µ
g/paw). Each measurement represented as mean
±
SEM of
n= 6
. Significantly different from VEH group (* p< 0.05) determined by ANOVA followed
by Dunnett’s post hoc test,
Figure S3
: Total ion chromatograms of volatile components from Bacharris
heterophylla obtained by extraction of HS-SPME,
Figure S4
: Representative HPLC chromatogram at
λ= 327 nm
, and UV absorption spectrum (200 to 400 nm) of compound
1
,
Figure S5
: Representative
HPLC chromatogram at
λ
= 327 nm, and UV absorption spectrum (200 to 400 nm) of compound
3
,
Figure S6:
Representative HPLC chromatogram at
λ
= 327 nm, and UV absorption spectrum (200
to 400 nm) of compound
4
,
Figure S7:
Calibration curve (area versus concentration) and residual
plot for compound
1
,
Figure S8:
Calibration curve (area versus concentration) and residual plot for
compound
3
,
Figure S9:
Calibration curve (area versus concentration) and residual plot for compound
4, Table S1. 1
H NMR and
13
C NMR spectral data of compounds
1−5
(400 and 100 MHz respectively;
MeOH-d
4
) from the aerial parts of Baccharis heterophylla,
Table S2. 1
H NMR and
13
C NMR spectral
data of compounds
6−8
(400 and 100 MHz respectively, DMSO-d
6
) from the aerial parts of Baccharis
heterophylla,Figure S10: EI-MS of volatile compounds 9–30 from Baccharis heterophylla.
Author Contributions:
Conceptualization, E.C.-R. and R.M.; methodology, E.C.-R., A.P.-V.,
R.T.-C.
,
A.N., A.A.-C. and R.M.; investigation, E.C.-R. and A.P.-V.; resources, A.N., A.A.-C. and R.M.; data cu-
ration, E.C.-R. and R.M.; writing—original draft preparation, E.C.-R., A.P.-V. and R.M.; writing—
review and editing, R.M.; visualization, E.C.-R.; supervision, A.N., A.A.-C. and R.M.; funding acqui-
sition, R.M. All authors have read and agreed to the published version of the manuscript.
Funding:
The research was supported by grants from CONACyT CB A1-S-11226 and DGAPA IN
217320 awarded to R.M.
Institutional Review Board Statement:
The experimental protocols were approved by the Institu-
tional Committee for Care and Use of Laboratory Animals (CICUAL-FQ) of Facultad de Química,
UNAM (FQ/CICUAL/391/19).
Informed Consent Statement: Not applicable.
Plants 2021,10, 116 12 of 13
Data Availability Statement: Not applicable.
Acknowledgments:
The authors recognize the valuable support of Georgina Duarte, Nayeli Lopez
Balbiaux and Marisela Gutiérrez Franco, from USAII-Facultad de Química UNAM for recording
NMR and Mass spectra. We are indebted to Christian Alan Cabello-Hernández (Facultad de Ciencias,
UNAM) for his valuable help for pursuing the animal experimentation. E.C.-R. acknowledges the
fellowship from CONACyT (257969) to pursue graduate studies.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Heinrich, M. Ethnobotany and its role in drug development. Phytother. Res. 2000,14, 479–488. [CrossRef]
2.
Navarro, R.C.; Sánchez, E.Y.P.; Maya, A.P. Aproximación crítica a las políticas públicas en salud indígena, medicina tradicional e
interculturalidad en México (1990–2016). Salud Colect. 2017,13, 443–455. [CrossRef] [PubMed]
3.
Kunle, O.F.; Egharevba, H.O.; Ahmadu, P.O. Standardization of herbal medicines—A review. Int. J. Biodivers. Conserv.
2012
,
4, 101–112. [CrossRef]
4.
Wagner, H.; Ulrich-Merzenich, G. Synergy research: Approaching a new generation of phytopharmaceuticals. Phytomedicine
2009
,
16, 97–110. [CrossRef]
5. Villaseñor, J.L. Checklist of the native vascular plants of Mexico. Rev. Mex. Biodivers. 2016,87, 559–902. [CrossRef]
6.
Rojas, A.; Bah, M.; Rojas, J.I.; Serrano, V.; Pacheco, S. Spasmolytic activity of some plants used by the Otomi Indians of Queretaro
(Mexico) for the treatment of gastrointestinal disorders. Phytomedicine 1999,6, 367–371. [CrossRef]
7. Abad, M.J.; Bermejo, P. Baccharis (Compositae): A review update. Arkivoc 2007,7, 76–96. [CrossRef]
8.
Bello-González, M.Á.; Hernández-Muñoz, S.; Lara-Chávez, M.; Nieves, B.; Salgado-Garciglia, R. Plantas útiles de la comunidad
indígena nuevo San Juan Parangaricutiro, Michoacán, México. Polibotánica 2015,39, 175–215. [CrossRef]
9.
Prada, J.; Ordúz-Díaz, L.L.; Coy-Barrera, E. Baccharis latifolia: Una Asteraceae poco valorada con potencialidad Química y
Biológica en el Neotrópico. Rev. Fac. Cienc. Bas. 2016,12, 92–105. [CrossRef]
10.
Martínez-López, J.; Acosta-Ramos, A.; Martínez-Ojeda, E.; Manzano-Méndez, F. Recursos forestales no maderables en dos
comunidades zapotecas de la Sierra Juárez de Oaxaca. Rev. Mex. Cienc. Forestales. 2016,7, 37–52. [CrossRef]
11.
Wollenweber, E.; Schober, I.; Dostal, P.; Hradetzky, D.; Arriaga-Giner, F.J.; Yatskievych, G. Flavonoids and terpenoids from the
exudates of some Baccharis species. Z. Naturforsch. C 1986,41, 87–93. [CrossRef]
12.
Arriaga-Giner, F.J.; Wollenweber, E.; Schober, I.; Dostal, P.; Braunt, S. 2
β
-Hydroxyhautriwaic acid, a clerodane type diterpenoid
and other terpenoids from three Baccharis species. Phytochemistry 1986,25, 719–721. [CrossRef]
13.
Jakupovic, J.; Schuster, A.; Ganzer, U.; Bohlmann, F.; Boldt, P.E. Sesqui-and diterpenes from Baccharis species. Phytochemistry
1990
,
29, 2217–2222. [CrossRef]
14.
Rojas, A.; Mendoza, S.; Moreno, J.; Arellano, R.O. Extracts from plants used in mexican traditional medicine activate Ca
2+
-
dependent chloride channels in Xenopus laevis oocytes. Phytomedicine 2003,10, 416–421. [CrossRef]
15.
Tjølsen, A.; Berge, O.G.; Hunskaar, S.; Rosland, J.H.; Hole, K. The formalin test: An evaluation of the method. Pain
1992
,51, 5–17.
[CrossRef]
16.
Pérez-Vásquez, A.; Aguilar-Cruz, R.; Bye, R.; Linares, E.; Rivero-Cruz, I. UHPLC-MS Analysis of Polyphenols in the Aqueous
Extract of Hydrangea seemannii.Rev. Bras. Farmacogn. 2020,30, 7–11. [CrossRef]
17.
Tamayose, C.I.; Torres, P.B.; Roque, N.; Ferreira, M.J.P. HIV-1 reverse transcriptase inhibitory activity of flavones and chlorogenic
acid derivatives from Moquiniastrum floribundum (Asteraceae). S. Afr. J. Bot. 2019,123, 142–146. [CrossRef]
18.
Silva, E.L.; Lobo, J.F.R.; Vinther, J.M.; Borges, R.M.; Staerk, D. High-Resolution
α
-Glucosidase Inhibition Profiling Combined with
HPLC-HRMS-SPE-NMR for Identification of Antidiabetic Compounds in Eremanthus crotonoides (Asteraceae). Molecules
2016
,
21, 782. [CrossRef]
19.
Van Loo, P.; De Bruyn, A.; Budˇešínský, M. Reinvestigation of the structural assignment of signals in the
1
H and
13
C NMR spectra
of the flavone apigenin. Magn. Reson. Chem. 1986,24, 879–882. [CrossRef]
20.
Miyazawa, M.; Hisama, M. Antimutagenic activity of flavonoids from Chrysanthemum morifolium.Biosci. Biotechnol. Biochem.
2003
,
67, 2091–2099. [CrossRef]
21.
Dos Santos, M.D.; Gobbo-Neto, L.; Albarella, L.; de Souza, G.E.P.; Lopes, N.P. Analgesic activity of di-caffeoylquinic acids from
roots of Lychnophora ericoides (Arnica da serra). J. Ethnopharmacol. 2005,96, 545–549. [CrossRef] [PubMed]
22.
Choi, M.Y.; Park, H.J. Antinocicepetive effects of 3, 4-Dicaffeoyl Quinic acid of Ligularia fischeri var. spiciformis.Korean J. Plant Res.
2007,20, 221–225.
23.
Dos Santos, M.D.; Almeida, M.C.; Lopes, N.P.; De Souza, G.E.P. Evaluation of the anti-inflammatory, analgesic and antipyretic
activities of the natural polyphenol chlorogenic acid. Biol. Pharm. Bull. 2006,29, 2236–2240. [CrossRef] [PubMed]
24.
Abdel Motaal, A.; Ezzat, S.M.; Tadros, M.G.; El-Askary, H.I.
In vivo
anti-inflammatory activity of caffeoylquinic acid derivatives
from Solidago virgaurea in rats. Pharm. Biol. 2016,54, 2864–2870. [CrossRef] [PubMed]
25.
Kim, Y.; Kim, J.T.; Park, J.; Son, H.J.; Kim, E.Y.; Lee, Y.J.; Rhyu, M.R. 4, 5-Di-O-Caffeoylquinic Acid fromLigularia fischeri Suppresses
Inflammatory Responses Through TRPV1 Activation. Phytother. Res. 2017,31, 1564–1570. [CrossRef] [PubMed]
Plants 2021,10, 116 13 of 13
26.
Hong, S.; Joo, T.; Jhoo, J.W. Antioxidant and anti-inflammatory activities of 3, 5-dicaffeoylquinic acid isolated from Ligularia fis-
cheri leaves. Food. Sci. Biotechnol. 2015,24, 257–263. [CrossRef]
27.
Wan, P.; Xie, M.; Chen, G.; Dai, Z.; Hu, B.; Zeng, X.; Sun, Y. Anti-inflammatory effects of dicaffeoylquinic acids from Ilex kudingcha
on lipopolysaccharide-treated RAW264. 7 macrophages and potential mechanisms. Food Chem. Toxicol.
2019
,126, 332–342.
[CrossRef] [PubMed]
28.
Tian, D.; Yang, Y.; Yu, M.; Han, Z.Z.; Wei, M.; Zhang, H.W.; Jia, H.M.; Zou, Z.M. Anti-inflammatory chemical constituents of
Flos Chrysanthemi Indici determined by UPLC-MS/MS integrated with network pharmacology. Food Funct.
2020
,11, 6340–6351.
[CrossRef]
29.
Xiao, X.; Wang, X.; Gui, X.; Chen, L.; Huang, B. Natural flavonoids as promising analgesic candidates: A systematic review.
Chem. Biodivers. 2016,13, 1427–1440. [CrossRef]
30.
Campos, F.R.; Bressan, J.; Jasinski, V.C.G.; Zuccolotto, T.; da Silva, L.E.; Cerqueira, L.B. Baccharis (Asteraceae): Chemical
constituents and biological activities. Chem. Biodivers. 2016,13, 1–17. [CrossRef]
31.
Dawidowicz, A.L.; Szewczyk, J.; Dybowski, M.P. Modified application of HS-SPME for quality evaluation of essential oil
plant materials. Talanta 2016,46, 195–202. [CrossRef] [PubMed]
32.
Liapi, C.; Anifantis, G.; Chinou, I.; Kourounakis, A.P.; Theodosopoulos, S.; Galanopoulou, P. Antinociceptive properties of 1,
8-cineole and
β
-pinene, from the essential oil of Eucalyptus camaldulensis leaves, in rodents. Planta Med.
2007
,73, 1247–1254.
[CrossRef] [PubMed]
33.
Kim, D.S.; Lee, H.J.; Jeon, Y.D.; Han, Y.H.; Kee, J.Y.; Kim, H.J.; Shin, H.J.; Kang, J.; Lee, B.S.; Kim, S.H.; et al. Alpha-pinene
exhibits anti-inflammatory activity through the suppression of MAPKs and the NF-
κ
B pathway in mouse peritoneal macrophages.
Am. J. Chin. Med. 2015,43, 731–742. [CrossRef] [PubMed]
34.
Paula-Freire, L.I.G.; Andersen, M.L.; Molska, G.R.; Köhn, D.O.; Carlini, E.L.A. Evaluation of the antinociceptive activity of
Ocimum gratissimum L. (Lamiaceae) essential oil and its isolated active principles in mice. Phytother. Res.
2013
,27, 1220–1224.
[CrossRef]
35.
Rufino, A.T.; Ribeiro, M.; Sousa, C.; Judas, F.; Salgueiro, L.; Cavaleiro, C.; Mendes, A.F. Evaluation of the anti-inflammatory, anti-
catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis.
Eur. J. Pharmacol.
2015,750, 141–150. [CrossRef]
36.
Vieira, A.J.; Beserra, F.P.; Souza, M.C.; Totti, B.M.; Rozza, A.L. Limonene: Aroma of innovation in health and disease.
Chem. Biol. Interact. 2018,283, 97–106. [CrossRef]
37.
International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human
Use (ICH): Q2 (R1): Validation of Analytical Procedures: Text and Methodology: Switzerland. Available online: http://academy.
gmp-compliance.org/guidemgr/files/Q2(R1).pdf (accessed on 9 December 2020).
38.
Reyes-Pérez, V.; Vásquez, A.P.; Déciga-Campos, M.; Bye, R.; Linares, E.; Mata, R. Antinociceptive Potential of Zinnia Grandiflora.
J. Nat. Prod. 2019,82, 456–461. [CrossRef]
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