![Tentative identification of compounds in the methanolic extract of S. cinnabarina, S. lavandu- loides y S. longispicata. Retention time (Rt) in minutes. (m/z) = mass/charge ratio. Mass error in ppm. [M + H] + , [M − H] − indicate the identification of compounds in positive and negative mode, respec- tively. S. cinnabarina (SCi), S. lavanduloides (SLa) y S. longispicata (SLo). n.i. = non-identified. The + symbol indicates the presence of compound.](https://www.researchgate.net/profile/Jose-Guerrero-Analco/publication/386005102/figure/tbl1/AS:11431281291753068@1732184322898/Tentative-identification-of-compounds-in-the-methanolic-extract-of-S-cinnabarina-S_Q320.jpg)
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Citation: Ortiz-Mendoza, N.;
Monribot-Villanueva, J.L.;
Guerrero-Analco, J.A.;
Martínez-Gordillo, M.J.; Basurto-Peña,
F.A.; Aguirre-Hernandez, E.;
Soto-Hernández, M. Comparative
Metabolomic Analysis and
Antinociceptive Effect of Methanolic
Extracts from Salvia cinnabarina,Salvia
lavanduloides and Salvia longispicata.
Molecules 2024,29, 5465. https://
doi.org/10.3390/molecules29225465
Academic Editor: Kemal Husnu
Can Baser
Received: 2 October 2024
Revised: 8 November 2024
Accepted: 18 November 2024
Published: 20 November 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Comparative Metabolomic Analysis and Antinociceptive Effect
of Methanolic Extracts from Salvia cinnabarina,Salvia
lavanduloides and Salvia longispicata
Nancy Ortiz-Mendoza 1,2 , Juan L. Monribot-Villanueva 3, JoséA. Guerrero-Analco 3,* ,
Martha J. Martínez-Gordillo 4, Francisco A. Basurto-Peña 5, Eva Aguirre-Hernandez 1, *
and Marcos Soto-Hernández 6
1Laboratorio de Productos Naturales, Departamento de Ecología y Recursos Naturales, Facultad de Ciencias,
Universidad Nacional Autónoma de México, Mexico City 04510, Mexico; nancy_om@ciencias.unam.mx
2Posgrado en Ciencias Biológicas, Unidad de Posgrado, Edificio D, 1◦Piso, Circuito de Posgrados, Ciudad
Universitaria Coyoacán, Mexico City 04510, Mexico
3Red de Estudios Moleculares Avanzados, Instituto de Ecología A. C., Carretera Antigua a Coatepec 351,
Xalapa 91073, Mexico; juan.monribot@inecol.mx
4
Departamento de Biología Comparada, Herbario de la Facultad de Ciencias, Universidad Nacional Autónoma
de México, Mexico City 04510, Mexico; mjmg@ciencias.unam.mx
5Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City 04510,
Mexico; abasurto@ib.unam.mx
6Posgrado en Botánica, Colegio de Postgraduados, Campus Montecillo, Texcoco 56264, Mexico;
msoto@colpos.mx
*Correspondence: joseantonio.guerrero@inecol.mx (J.A.G.-A.); eva_aguirre@ciencias.unam.mx (E.A.-H.)
Abstract: Mexico is considered one of the countries with the greatest diversity of the Salvia genus.
A significant percentage of its species are known for their use in traditional medicine, highlighting
their use as an analgesic. The objective of this work was to determine the chemical composition
of the methanolic extracts of S. cinnabarina,S. lavanduloides and S. longispicata through untargeted
metabolomics, as well as the
in vivo
evaluation of the antinociceptive effect and acute oral toxicity. The
chemical profiling was performed using ultra-high performance liquid chromatography coupled with
a high-resolution mass spectrometry (UPLC-ESI
+/−
-MS-QTOF) system and tentative identifications
were performed using a compendium of information on compounds previously isolated from Mexican
species of the genus. Pharmacological evaluation was carried out using the formalin test and OECD
guidelines. The analysis of the spectrometric features of the mass/charge ratios of the three salvias
shows that a low percentage of similarity is shared between them. Likewise, the putative identification
allowed the annotation of 46 compounds, mainly of diterpene and phenolic nature, with only four
compounds shared between the three species. Additionally, the extracts of the three salvias produced
a significant antinociceptive effect at a dose of 300 mg/kg administered orally and did not present an
acute oral toxicity effect at the maximum dose tested, indicating a parameter of
LD50 > 2000 mg/kg.
The exploration of the chemical profile of the three salvias by untargeted metabolomics shows
that, despite being species with antinociceptive potential, they have different chemical profiles and
therefore different active metabolites.
Keywords: antinociceptive effect; Salvia; terpenoids; flavonoids; UPLC-MS
1. Introduction
The species of the genus Salvia L. have been used throughout the world for their
broad spectrum of biological activities, to name a few, they have been used for the treat-
ment of digestive problems, cardiovascular and cerebrovascular diseases, pain, bronchitis,
cough, asthma, inflammation, depression, anxiety, insomnia, and skin conditions [
1
–
4
].
Pharmacological studies report its antioxidant, antidiabetic, antiviral, antinociceptive,
Molecules 2024,29, 5465. https://doi.org/10.3390/molecules29225465 https://www.mdpi.com/journal/molecules
Molecules 2024,29, 5465 2 of 18
anti-inflammatory, anti-Alzheimer, and antitumor properties, among others. Terpenoids,
phenolic acids, and flavonoids are compounds responsible for the medicinal effects of
sage [
2
–
8
]. Regarding the analgesic properties of Salvia, there is evidence in the literature
of its effect through different models of nociceptive and inflammatory pain in rodents.
The ethanolic extract of S. plebeia R.Br. presents pharmacological activity in the abdominal
pain model and in the inflammatory process induced in the carrageenan test [
9
]. The
hydroalcoholic extract of S. officinalis L. produces antinociceptive and anti-inflammatory
effects in abdominal stretching, formalin and carrageenan tests [
10
]. Likewise, the hydroal-
coholic extract of S. miltiorrhiza Bunge has analgesic and anti-inflammatory effects using
the collagen-induced arthritis model [7]. Similarly, the ethanolic extract of S. lachnostachys
Benth. had good activity both in the formalin test and the anti-arthritis model [11].
Mexico is considered one of the areas with the greatest diversity of the Salvia genus
in the world, represented by around 307 species [
12
]. Almost all Mexican salvias (sages)
species are included within the subgenus Calosphace [
13
,
14
]. From ancient times to the
present, various species of Salvia have been known for their wide range of ornamental,
cosmetic, culinary and medicinal uses [
15
–
18
]. The last one is the most notable, as there
are around 56 species belonging to the subgenus Calosphace used in traditional Mexican
medicine to treat various diseases of the digestive system, nervous disorders, pregnancy,
childbirth and postpartum, and other culture-bound syndromes [
19
]. Within the wide
spectrum of properties of traditionally used Mexican salvias, analgesic stands out [
16
,
19
,
20
].
Some examples of sages with this characteristic are S. microphylla Kunth, S. coccinea Buc’hoz
ex Etl., S. lavanduloides Kunth, S. elegans Vahl, S. polystachia Cav., S. leucantha Cav., S. mexi-
cana L., S. hispanica L., S. amarissima Ortega, and S. tiliifolia Vahl, of which the aerial part
is used, prepared as an infusion, to treat pain, mainly in south-central Mexico (Yucatan,
Chiapas, Oaxaca, Guerrero, Morelos, Michoacan) [
21
–
30
]. Despite the analgesic proper-
ties of Mexican salvias, most
in vitro
,
in vivo
and ex vivo studies, both of extracts and
isolated compounds, have been mainly directed to the evaluation of antimicrobial and
cytotoxic effects [
31
–
33
]. Only the species S. amarissima (Syn. S. circinate Cav.), S. purpurea
Cav., S. semiatrata Zucc., and S. tiliifolia of the subgenus Calosphace have been evaluated in
antinociception models. Studies have reported no acute oral toxicity of the extracts and a
significant reduction in nociception of extracts of different polarity, at doses between 100
and 300 mg/kg, administered orally (p.o.), as well as of isolated compounds of diterpene
nature (amarisolide A, tilifodiolide and 7-keto-neoclerodan-3,13-dien-18,19:15,16-diolide)
and phenolic (pedalitin) at doses of 1–10 mg/kg, p.o. [
34
–
37
]. The potent antinociceptive
effect of compounds of this nature, isolated from other botanical families, has been cor-
roborated in
in vivo
tests [
37
–
40
]. In relation to the above, it should be noted that various
phytochemical studies of approximately 50 species of Salvia of the subgenus Calosphace
have reported the presence of secondary metabolites of terpene and phenolic nature, with
diterpenes (abietane, clerodane, labdane and pimarane) being the most abundant, even
postulated as chemotaxonomic markers [
1
,
19
,
41
,
42
]. S. cinnabarina M.Martens & Galeotii, S.
lavanduloides and S. longispicata M.Martens & Galeotii, are three species of the subgenus
Calosphace, used in tea form for analgesic purposes in traditional Mexican medicine and
that have a wide distribution in Mexican territory, with the exception of the Baja California
peninsula [
43
]. There are no reports on the antinociceptive potential in
in vivo
models and
little is known about its phytochemistry. Therefore, in this study, the chemical composition
is explored by means of an untargeted metabolomic analysis, using ultra-high performance
liquid chromatography coupled with high-resolution mass spectrometry (UPLC-ESI
+/−
-
MS-QTOF), for the identification of a significant number of specialised metabolites in the
selected species. Likewise, acute oral toxicity is evaluated following the Organisation for
Economic Co-operation and Development (OECD) Guidelines for the Testing of Chemicals
(2001), and antinociceptive activity using the formalin test of methanol extract by oral
route [44].
Molecules 2024,29, 5465 3 of 18
2. Results
2.1. Chemical Profiling via Ultra Performance Liquid Chromatography Coupled to Mass
Spectrometry Quadrupole Time of Flight (UPLC-ESI+/−-MS-QTOF)
2.1.1. Preliminary Comparison of the Retention Time-Mass/Charge Features Between
Salvia Species
A preliminary comparative analysis of the constitution of retention time-mass/charge
(rt-m/z) features obtained by UPLC-ESI
+/−
-MS-QTOF, of the three sages, shows that they
share a low percentage of similarity between them. This is visualised by the Venn diagram
(Figure 1A) and the principal component analysis (PCA) (Figure 1B), which indicate that
the three salvias do not group together by similarity in their rt-m/zprofile.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 19
2. Results
2.1. Chemical Profiling via Ultra Performance Liquid Chromatography Coupled to Mass
Spectrometry Quadrupole Time of Flight (UPLC-ESI+/−-MS-QTOF).
2.1.1. Preliminary Comparison of the Retention Time-Mass/Charge Features Between
Salvia Species
A preliminary comparative analysis of the constitution of retention time-mass/charge
(rt-m/z) features obtained by UPLC-ESI+/−-MS-QTOF, of the three sages, shows that they
share a low percentage of similarity between them. This is visualised by the Venn diagram
(Figure 1A) and the principal component analysis (PCA) (Figure 1B), which indicate that
the three salvias do not group together by similarity in their rt-m/z profile.
Figure 1. Comparative analysis by Venn diagram (A) and PCA (B) of the constitution of the rt-m/z
features of the methanolic extracts of S. cinnabarina, S. lavanduloides and S. longispicata obtained by
UPLC-ESI+/−-MS-QTOF.
2.1.2. Difference in Chemical Composition of Sages
The experimental m/z values obtained from the three sages compared with the data
reported from molecules previously isolated from Mexican Salvia species allowed the ten-
tative identification of 46 metabolites including five phenolic acids, 13 flavonoids, 24 diter-
penes and four triterpenes. Table 1 shows the chromatographic (rt) and spectrometric
(m/z) data for each compound.
Table 1. Tentative identification of compounds in the methanolic extract of S. cinnabarina, S. lavandu-
loides y S. longispicata. Retention time (Rt) in minutes. (m/z) = mass/charge ratio. Mass error in ppm.
[M + H]+, [M − H]− indicate the identification of compounds in positive and negative mode, respec-
tively. S. cinnabarina (SCi), S. lavanduloides (SLa) y S. longispicata (SLo). n.i. = non-identified. The +
symbol indicates the presence of compound.
No. Rt (min) (m/z) Compound Adduct
Mass
Error
(ppm)
SCi SLa SLo Reference
1 0.52 719.1643 Sagerinic acid [M − H]− 4.3 + n.i n.i [45]
2 2.74 563.1403 Schaftoside [M − H]− 0.4 n.i + n.i [46]
3 3.41 197.0449 Syringic acid [M − H]− −0.5 n.i n.i + [47]
4 3.91 610.1548 Cyanidin 3,5-diglucoside [M − H]− 2.3 n.i n.i + [48]
5 4.21 609.1461 Rutin [M − H]− 0.8 + + + [47]
6 4.29 477.0678 Miquelianin [M − H]− 1.9 n.i n.i + [46]
7 4.33 463.0881 Quercetin-O-hexoside [M − H]− 0.8 n.i + + [5]
Figure 1. Comparative analysis by Venn diagram (A) and PCA (B) of the constitution of the rt-m/z
features of the methanolic extracts of S. cinnabarina,S. lavanduloides and S. longispicata obtained by
UPLC-ESI+/−-MS-QTOF.
2.1.2. Difference in Chemical Composition of Sages
The experimental m/zvalues obtained from the three sages compared with the data re-
ported from molecules previously isolated from Mexican Salvia species allowed the tentative
identification of 46 metabolites including five phenolic acids, 13 flavonoids, 24 diterpenes
and four triterpenes. Table 1shows the chromatographic (rt) and spectrometric (m/z) data
for each compound.
Table 1. Tentative identification of compounds in the methanolic extract of S. cinnabarina,S. lavan-
duloides yS. longispicata. Retention time (Rt) in minutes. (m/z) = mass/charge ratio. Mass error in
ppm. [M + H]
+
, [M
−
H]
−
indicate the identification of compounds in positive and negative mode,
respectively. S. cinnabarina (SCi), S. lavanduloides (SLa) y S. longispicata (SLo). n.i. = non-identified.
The + symbol indicates the presence of compound.
No. Rt (min) (m/z) Compound Adduct
Mass
Error
(ppm)
SCi SLa SLo Reference
1 0.52 719.1643 Sagerinic acid [M −H]−4.3 + n.i n.i [45]
2 2.74 563.1403 Schaftoside [M −H]−0.4 n.i + n.i [46]
3 3.41 197.0449 Syringic acid [M −H]−−0.5 n.i n.i + [47]
4 3.91 610.1548 Cyanidin 3,5-diglucoside [M −H]−2.3 n.i n.i + [48]
5 4.21 609.1461 Rutin [M −H]−0.8 + + + [47]
6 4.29 477.0678 Miquelianin [M −H]−1.9 n.i n.i + [46]
7 4.33 463.0881 Quercetin-O-hexoside [M −H]−0.8 n.i + + [5]
8 4.46 461.0725 Luteolin-7-O-glucuronide [M −H]−1.1 + n.i n.i [45]
9 4.67 521.1296 Salviaflaside [M −H]−0.2 + n.i + [45]
Molecules 2024,29, 5465 4 of 18
Table 1. Cont.
No. Rt (min) (m/z) Compound Adduct
Mass
Error
(ppm)
SCi SLa SLo Reference
10 4.72 477.1045 Rhamnetin 3-glucoside [M −H]−2.5 + + n.i [46]
11 4.79 301.0344 Quercetin [M −H]−−1.3 n.i + n.i [34]
12 5.08 269.0448 Apigenin [M −H]−−0.7 + n.i n.i [49]
13 5.20 359.0775 Rosmarinic acid [M −H]−2.2 + + + [45]
14 5.37 493.1141 Salvianolic acid A [M −H]−1.2 n.i + n.i [45]
15 5.39 285.0397 Luteolin [M −H]−−0.7 + + n.i [45]
16 5.60 315.0510 Pedalitin [M −H]−1.6 n.i + + [50]
17 5.92 343.0816 5,6-Dihydroxy-7,3’,4’-
trimethoxyflavone [M −H]−−0.6 + n.i n.i [49]
18 6.22 287.0554 Kaempferol [M + H]+−0.7 + n.i n.i [51]
6.22 285.0398 Kaempferol [M −H]−−0.3 n.i + n.i. [51]
19 6.54 593.1865 Amarisolide F [M −H]−−0.8 n.i + + [49]
20 6.74 355.1182 Dugesin G [M −H]−0.0 n.i + n.i. [52]
21 6.75 337.1092 Salvixalapadiene [M −H]−4.7 n.i + + [53]
22 6.81 349.2008 2α,6α-Dihydroxy-patagonol [M −H]−−2.0 n.i + n.i [54]
23 6.90 347.1863 15,16-Diol-15,16-dihydro-
hardwickiic acid [M −H]−1.4 + n.i n.i [55]
24 7.03 357.1333 Splenolide A [M −H]−−1.4 n.i + n.i [56]
25 7.20 371.1132 Salvianduline B [M −H]−0.3 n.i + n.i [57]
26 8.14 399.1435 Salvimexicanolide [M −H]−−2.2 n.i + n.i [58]
27 8.17 347.1864 16-Hydroxycarnosic acid [M −H]−1.7 n.i + + [59]
28 8.20 345.1711 16-Hydroxycarnosol [M −H]−2.6 n.i + [60]
29 8.30 333.2066 11,12,16,20-Tetrahydroxy-
abieta-8,11,13-triene [M −H]−0.0 + n.i n.i [59]
30 9.23 315.1954 20-Deoxocarnosol [M −H]−−1.9 + n.i n.i [59]
31 9.91 389.1961 16-Acetoxycarnosic acid [M −H]−−0.8 + n.i [59]
32 9.94 329.1752 Carnosol [M −H]−−0.3 + n.i n.i [5]
33 10.10 331.1909 Carnosic acid [M −H]−0.0 + n.i n.i [59]
34 10.39 331.1914 5,6-Dihydro-6α-
hydroxysalviasperanol [M −H]−1.5 + n.i + [61]
35 10.58 329.1757 19-Deoxyicetexone [M]−1.2 n.i n.i + [62]
36 10.66 317.2120 Demethylsalvicanol [M −H]−0.9 + n.i n.i [61]
37 11.33 389.1971 7α-Acetoxy-6,7-
dihydroicetexone [M]−1.8 n.i + n.i [41]
38 11.42 301.1809 Royleanone [M −H]−1.6 n.i n.i + [63]
39 11.43 343.1549 Rosmadial [M −H]−1.2 n.i n.i + [60]
40 11.47 355.1542 Tilifolidione [M −H]−−1.1 n.i n.i + [63]
41 11.63 301.2170 3,4-Secoisopimar-4(18),7,15-
trien-3-oic acid [M −H]−0.7 + n.i n.i [64]
42 12.16 317.2110 6β-Hydroxy-trans-communic
acid [M −H]−−2.2 + n.i n.i [65]
43 12.21 455.3161 Dihydroxy-24-nor-4(23),12-
oleanadien-28-oic acid [M −H]−0.0 + n.i n.i [66]
44 12.34 275.2006 3-Hydroxyestran-17-one [M −H]−−1.8 + + + [67]
45 13.29 469.3325 11β-Hydroxy-3-oxo-urs-12-en-
28-oic acid [M −H]−1.5 + + + [68]
46 13.44 471.3481 2-Hydroxyursolic acid [M −H]−1.5 + n.i + [69]
Molecules 2024,29, 5465 5 of 18
It is important to note that when comparing the historical data of compounds pre-
viously isolated from Mexican salvias with the experimental data, only 46 data points
were correlated with a mass error of less than 5 ppm in negative mode and one in positive
mode. The positive value corresponds to kaempferol, which is also identified in negative
mode. Both are indicated as compound 18 with the following notation: Kaempferol*,
in positive mode, was identified in S. cinnabarina and Kaempferol**, in negative mode,
in S. lavanduloides.
The most relevant results are briefly mentioned. Diterpenes were the group with
the highest number of molecules identified in the extracts of sages (Table 1, No. 19–42),
detected in rt from 6.54 to 12.16 min. The abietanes and clerodanes stand out with 14 and
8 structures,
respectively. The abietanes (29,30,32–34,36), clerodane 23, pimarane 41 and
labdane 42 were identified in S. cinnabarina. The abietanes (27,28,34,35,38–40) and the
clerodanes, amarisolide F (19) and salvixalapadiene (21) were present in S. longispicata.
Furthermore, seven clerodanes (19–22,24–26) and three abietanes (27,31,37) were detected
in S. lavanduloides, identified as 16-hydroxycarnosic acid, 16-acetoxycarnosic acid and 7
α
-
acetoxy-6,7 dihydroicetexone, respectively. Flavonoids were the second largest group with
13 compounds identified (2,4–8,10–12,15–18). Rutin (5) was present in all three salvias,
whereas luteolin-7-O-glucuronide (8), apigenin (12) and 5,6-dihydroxy-7,3
′
,4
′
-trimethoxy
(17) were only present in S. cinnabarina. Schaftoside (2), quercetin (11) and kaempferol
(18) were found in S. lavanduloides. Also, cyanidin 3,5-diglucoside (4) and miquelianin
(6) were identified only in S. longispicata. Phenolic acids were the third group identified
(1,3,9,13 and 14) at a retention time of 0.52, 3.41, 4.67, 5.2 and 5.37 min, respectively.
Compound 1, identified as sagerinic acid, represented by the molecular ion m/z719.1643,
was detected only in S. cinnabarina. Syringic acid (3) with an ion m/zat 197.0449 was
found in S. longispicata and salvianolic acid A (14) with m/zat 493.1141 in S. lavanduloides.
Rosmarinic acid (13) with an ion m/zat 359.0775 was present in all three salvias. Finally,
triterpenoids were identified as a fourth group, an oleanane derivative (43) was only present
in S. cinnabarina, while 3-hydroxyestran-17-one (44) and an ursane derivative (45) were
found in all three salvias (Table 1).
The chemical structures of the identified compounds are shown in Figure 2A. The
comparative analysis of the chemical composition of the three sages using a Venn diagram
(Figure 2B) showed that S. lavanduloides shares more compounds with S. longispicata (7,16,
19,21,27), corresponding to quercetin glycoside, pedalitin, amarisolide F, salvixalapadi-
ene, and 16-hydroxycarnosic acid, respectively. Three metabolites are common between
S. cinnabarina and S. longispicata: salviaflaside (9), 5,6-dihydro-6
α
-hydroxysalviasperanol
(34) and 2-hydroxyursolic acid (46). Likewise, rhamnetin 3-glucoside (10), luteolin (15)
and kaempferol (18) were identified in S. cinnabarina and S. lavanduloides. Of these com-
pounds, only four are shared between the three species: rutin (5), rosmarinic acid (13),
3-hydroxyestran-17-one (44), and 11
β
-hydroxy-3-oxo-urs-12-en-28-oic acid (45) (Figure 2B).
In S. cinnabarina, 13 chemical constituents were identified as unique (1,8,12,17,23,29,30,
32,33,36,41,42,43), 10 in S. lavanduloides (2,11,14,20,22,24,25,26,31,37), and 8 in S.
longispicata (3,4,6,28,35,38,39,40) (Figure 2B).
The differences of the 46 compounds between the three salvias are presented in a
heat map (Figure 3). In this map, red indicates a relatively higher intensity of each of
the compounds present in the sages and blue a lower intensity. Abietanes (27–40) and
clerodanes (19–26) were the most common among salvias, with 22 compounds identified.
Abietanes predominate in S. longispicata and S. cinnabarina, and clerodanes in S. lavandu-
loides. Pimarane (41) and labdane (42) were found in S. cinnabarina. Regarding phenolic
compounds (1–18) and triterpenoids (43–46), they are present in greater quantities in S.
cinnabarina and S. lavanduloides, compared to S. longispicata (Figure 3).
Molecules 2024,29, 5465 6 of 18
Molecules 2024,29, x FOR PEER REVIEW 6of 19
Figure 2. Metabolites present in the analysed Salvia species. The coloured blocks show the chemical
structures (A). The Venn diagram (B) shows the distribution of the compounds shared between the
species.
Figure 2. Metabolites present in the analysed Salvia species. The coloured blocks show the chemical
structures (A). The Venn diagram (B) shows the distribution of the compounds shared between
the species.
Molecules 2024,29, 5465 7 of 18
Molecules 2024,29, x FOR PEER REVIEW 7of 19
The differences of the 46 compounds between the three salvias are presented in a heat
map (Figure 3). In this map, red indicates a relatively higher intensity of each of the com-
pounds present in the sages and blue a lower intensity. Abietanes (27–40) and clerodanes
(19–26) were the most common among salvias, with 22 compounds identified. Abietanes
predominate in S. longispicata and S. cinnabarina, and clerodanes in S. lavanduloides.
Pimarane (41) and labdane (42) were found in S. cinnabarina. Regarding phenolic com-
pounds (1–18) and triterpenoids (43–46), they are present in greater quantities in S. cin-
nabarina and S. lavanduloides, compared to S. longispicata (Figure 3).
Figure 3. Comparative composition of S. cinnabarina, S. lavanduloides, and S. longispicata chemical
profiles. Clustered heat map analysis of 46 metabolites. Kaempferol*: detected in positive mode.
Kaempferol**: Detected in negative mode.
Figure 3. Comparative composition of S. cinnabarina,S. lavanduloides, and S. longispicata chemical
profiles. Clustered heat map analysis of 46 metabolites. Kaempferol*: detected in positive mode.
Kaempferol**: Detected in negative mode.
2.2. Acute Toxicity of the Methanol Extracts of Salvias
The methanolic extracts of the three sages did not produce acute toxicity effects at
the doses tested, nor at the maximum dose explored according to OECD’s test No. 423,
indicating a parameter of LD
50
> 2000 mg/kg, p.o. There was no significant difference in the
weight of mice receiving the extract compared to the vehicle during the 14-day evaluation
(Figure 4). Likewise, during the periodic observation throughout the assessment, no signs
of toxicity such as changes in the skin and fur, somatomotor activity or behavioural changes
were observed, nor were tremors, convulsions, diarrhoea, lethargy, sleep, or coma detected.
Molecules 2024,29, 5465 8 of 18
Molecules 2024, 29, x FOR PEER REVIEW 8 of 19
2.2. Acute Toxicity of the Methanol Extracts of Salvias
The methanolic extracts of the three sages did not produce acute toxicity effects at the
doses tested, nor at the maximum dose explored according to OECD’s test No. 423, indi-
cating a parameter of LD50 > 2000 mg/kg, p.o. There was no significant difference in the
weight of mice receiving the extract compared to the vehicle during the 14-day evaluation
(Figure 4). Likewise, during the periodic observation throughout the assessment, no signs
of toxicity such as changes in the skin and fur, somatomotor activity or behavioural
changes were observed, nor were tremors, convulsions, diarrhoea, lethargy, sleep, or
coma detected.
Figure 4. Time course of weight gain. Weight of mice recorded over 14 days in the assessment of
acute oral toxicity of S. cinnabarina, S. lavanduloides and S. longispicata extracts. Lines represent the
mean plus the Standard Error of the Mean (S.E.M.) of three animals, two-way ANOVA followed by
Dunne’s test.
2.3. Antinociceptive Effect of Sage Extracts on the Neurogenic and Inflammatory Phases of the
Formaline Test
Methanolic extracts at a dose of 300 mg/kg, p.o. and diclofenac (DFC, reference drug
at 10 mg/kg, p.o.) significantly reduced nociceptive behaviour in both the neurogenic
phase (F4,25 = 7.571, p < 0.0004) (Figure 5A) and the inflammatory phase (F4,25 = 19.93, p <
0.0001) (Figure 5B) compared to the group receiving the vehicle. However, the S. longispi-
cata extract showed a strong decrease in nociceptive behaviour in both phases, similar to
the reference drug.
Figure 5. Antinociceptive-like effects of methanol extracts and the reference drug (DCF, 10 mg/kg,
p.o.) in the nociceptive response of time spent in licking in the neurogenic (A) and inflammatory (B)
Figure 4. Time course of weight gain. Weight of mice recorded over 14 days in the assessment of
acute oral toxicity of S. cinnabarina,S. lavanduloides and S. longispicata extracts. Lines represent the
mean plus the Standard Error of the Mean (S.E.M.) of three animals, two-way ANOVA followed by
Dunnett’s test.
2.3. Antinociceptive Effect of Sage Extracts on the Neurogenic and Inflammatory Phases of the
Formaline Test
Methanolic extracts at a dose of 300 mg/kg, p.o. and diclofenac (DFC, reference drug
at 10 mg/kg, p.o.) significantly reduced nociceptive behaviour in both the neurogenic phase
(F
4,25
= 7.571, p< 0.0004) (Figure 5A) and the inflammatory phase (F
4,25
= 19.93,
p< 0.0001)
(Figure 5B) compared to the group receiving the vehicle. However, the S. longispicata
extract showed a strong decrease in nociceptive behaviour in both phases, similar to the
reference drug.
Molecules 2024, 29, x FOR PEER REVIEW 8 of 19
2.2. Acute Toxicity of the Methanol Extracts of Salvias
The methanolic extracts of the three sages did not produce acute toxicity effects at the
doses tested, nor at the maximum dose explored according to OECD’s test No. 423, indi-
cating a parameter of LD50 > 2000 mg/kg, p.o. There was no significant difference in the
weight of mice receiving the extract compared to the vehicle during the 14-day evaluation
(Figure 4). Likewise, during the periodic observation throughout the assessment, no signs
of toxicity such as changes in the skin and fur, somatomotor activity or behavioural
changes were observed, nor were tremors, convulsions, diarrhoea, lethargy, sleep, or
coma detected.
Figure 4. Time course of weight gain. Weight of mice recorded over 14 days in the assessment of
acute oral toxicity of S. cinnabarina, S. lavanduloides and S. longispicata extracts. Lines represent the
mean plus the Standard Error of the Mean (S.E.M.) of three animals, two-way ANOVA followed by
Dunne’s test.
2.3. Antinociceptive Effect of Sage Extracts on the Neurogenic and Inflammatory Phases of the
Formaline Test
Methanolic extracts at a dose of 300 mg/kg, p.o. and diclofenac (DFC, reference drug
at 10 mg/kg, p.o.) significantly reduced nociceptive behaviour in both the neurogenic
phase (F4,25 = 7.571, p < 0.0004) (Figure 5A) and the inflammatory phase (F4,25 = 19.93, p <
0.0001) (Figure 5B) compared to the group receiving the vehicle. However, the S. longispi-
cata extract showed a strong decrease in nociceptive behaviour in both phases, similar to
the reference drug.
Figure 5. Antinociceptive-like effects of methanol extracts and the reference drug (DCF, 10 mg/kg,
p.o.) in the nociceptive response of time spent in licking in the neurogenic (A) and inflammatory (B)
Figure 5. Antinociceptive-like effects of methanol extracts and the reference drug (DCF, 10 mg/kg,
p.o.) in the nociceptive response of time spent in licking in the neurogenic (A) and inflammatory (B)
phases after intraplantar injection of 20
µ
L of 1% formalin in mice. ANOVA followed by Dunnett’s
post hoc test. * p< 0.001 indicates significant difference in comparison to the vehicle group (SS).
3. Discussion
Salvia is the most diverse genus within the Lamiaceae family, and Mexico is home to
the largest number of species, with approximately 307 [
12
,
70
], of which around 56 species
have been used in traditional Mexican medicine, with different healing properties [
12
,
19
,
20
].
Despite the richness of the genus and its wide use for its medicinal properties in different
parts of the world [
1
,
19
,
71
,
72
], metabolomic studies are still few, mainly focused on species
with some use and addressing different objectives, such as S. miltiorrhiza from the Old World,
from the subgenus Glutinaria, or S. hispanica, from Mexico, which is within the subgenus
Calosphace [
73
]. This work includes three species of the subgenus Calosphace, which belong
to different sections and to different clades [
74
,
75
]. To explain the medicinal properties
Molecules 2024,29, 5465 9 of 18
of these species, in particular their antinociceptive effect, their chemical compositions
were obtained and compared with each other. Untargeted metabolomic analysis with
UPLC-ESI
+/−
-MS-QTOF allowed the identification of a total of 46 compounds of phenolic
and terpene nature in the extracts of salvias and also highlighted that the three species
present particular chemical profiles, with only four shared compounds (rutin, rosmarinic
acid, 11
β
-hydroxy-3-oxo-urs-12-en-28-oic acid and 3-hydroxyestran-17-one). The first three
are widely distributed, both in Salvia [
8
,
76
–
78
] and in various botanical families [
79
–
81
],
and this is explained because they are compounds that intervene in defence mechanisms
against other organisms or that improve tolerance to certain environmental factors such
as pollution, UV light and lack of water [
82
–
86
]. Phenolic acids such as sagerinic, syringic
and salvianolic were first identified in S. cinnabarina,S. longispicata, and S. lavanduloides,
respectively. These metabolites have been previously reported in Salvia species [
5
,
8
,
87
].
Regarding flavonoids, 13 were identified, some of which are exclusive to each sage such
as schaftoside, miquelianin, and luteolin-7-O-glucoronide detected in S. lavanduloides,S.
longispicata and S. cinnabarina, respectively. Common flavonoids of the genus were also
identified, like kaemperol, pedalitin, apigenin, quercetin, luteolin, and the glycosides of
the last three compounds [
5
,
8
,
87
]. With respect to triterpenoids, four compounds were
identified in the three salvias. Previous research reports that oleanolic acid, ursolic acid,
and their derivatives are common and present in almost all Salvia species [1,87–89].
Mainly terpene components are known in both S. cinnabarina and S. lavanduloides.
This work contributed to expanding the phytochemical knowledge of these species by
identifying the presence of structures from the group of phenolic compounds, which little
is known in Mexican sages. On the other hand, it is the first time that the metabolite profile
in S. longispicata has been investigated.
The most characteristic metabolites found in Salvia are the diterpenes (clerodanes and
abietanes). Clerodanes are almost restricted to Neotropical salvias and are found mainly
in the subgenus Calosphace. On the other hand, abietanes are present in European, Asian
and American sages, in all subgenera, and are also found in Mexican salvias, both in the
subgenus Audibertia, where they seem to be more abundant, and in the subgenus Calosphace,
to which the three species studied belong. In addition, 14 abietane and eight clerodane
structures were identified in this research. It should be noted that abietanes predominate
in S. longispicata and S. cinnabarina, and clerodanes in S. lavanduloides, in the latter, two
clerodanes have previously been isolated [
90
]. At the same time, this work reports, for the
first time, the presence of five more clerodanes and two more abietanes in S. lavanduloides.
On the other hand, five abietanes, one clerodane, one labdane, and one pimarane were
identified in the extract of S. cinnabarina. These last two compounds have already been
previously identified and isolated in this species [
64
,
65
]. It is worth noting that labdanes
and pimaranes are not very abundant in Salvia, being identified only in S. hispanica,S. parryi
A.Gray, S. fulgens Cav., S. microphylla,S. greggii A. Gray, S. sclarea L., and S. officinalis [1].
Specifically, the chemical profile of the three sages is contrasting, given that they are not
phylogenetically close and, despite the fact that they were all collected in the State of Oaxaca,
the microenvironments where they develop are different. Salvia cinnabarina belongs to the
Incarnatae section and is the only one that synthesizes pimaranes and labdanes [
64
,
65
,
91
],
compounds not present in the Lavanduloideae section and the Angulatae section, which
respectively include S. lavanduloides and S. longispicata, so these diterpenoids could be good
chemotaxonomic markers.
Flavonoids are common and widely distributed in angiosperms, also in the Lami-
aceae family and particularly in Salvia [
92
]. Chemotaxonomy has always been a field of
exploration, so it is not uncommon for phenolic compounds to be studied in this context,
investigating their usefulness in characterising and differentiating Taxa or in developing
hypotheses of phytochemical evolution [
93
], in addition to finding better species to obtain
natural products. Although it is not an objective of the work, in the case of flavonoids,
the comparison exercise is carried out between the three species, to investigate their value
as taxonomic markers. They are documented to present different profiles: S. cinnaba-
Molecules 2024,29, 5465 10 of 18
rina, a herb with red flowers and exserted stamens, exhibits seven flavonoids, of which
it shares four with S. lavanduloides, a herb with blue flowers and inset stamens, and one
with S. longispicata, a suffrutex with blue flowers and inset stamens. S. longispicata has four
flavonoids, of which it shares one with S. cinnabarina and two with S. lavanduloides. The
greatest similarity is found between S. cinnabarina and S. lavanduloides, something that is
not expected, given the morphological characteristics of each species and the position they
present in the phylogenies [
75
,
93
,
94
] since S. cinnabarina is located in a more basal clade
with respect to the other two, which are found in more recent and closer clades. We do not
consider these results sufficient to evaluate the value of these compounds as taxonomic
markers, and we think it is necessary to include a larger number of species in studies with
this objective; taking into account that, unlike Old World salvias, studies reporting phenolic
compounds for American sages are still scarce.
In order to determine the analgesic potential of Salvia species, the methanolic extracts
of the three salvias were evaluated using the formalin test. The results show the effect of
the extracts at the central and peripheral level, associated with the reduction of nociceptive
behaviour in the neurogenic and inflammatory phases.
It is worth noting that the extract of S. longispicata reduced nociceptive behavior to
a greater extent in both phases. This was possibly due to the chemical differences with
respect to the other two salvias. The main difference lies in the presence of a greater number
of abietane-type diterpenes. In this regard, the therapeutic effect of tanshinones, carnosic
acid and carnosol, isolated from various species of Salvia, is reported in various conditions
that cause nociceptive pain, associated with inflammation [38,95–97].
These results are consistent with the antinociceptive effect observed in other Salvia
species, such as the ethanolic extract and nor-abietane fruticulin, obtained from S. lach-
nostachys, that had good activity in the formalin test [
98
]. Additionally, the hydroal-
coholic extract and terpenoids isolated from S. officinalis produced antinociceptive and
anti-inflammatory activity in the Writhing, formalin, and carrageenan tests [
38
]. Extracts
of different polarity, as well as clerodane-type compounds from S. amarissima (Syn. Salvia
circinata), S. divinorum Epling & Jativa, S. semiatrata,S. purpurea, and S. tiliifolia promoted
significant effects in pain and inflammation tests (Writhing, formalin, hot plate, carrageenan
tests, and in a model of fibromyalgia and allodynia) [6,34–37,99,100].
Several studies have reported the analgesic and anti-inflammatory properties, both
in vitro
and
in vivo
models, of flavonoids such as apigenin, kaempferol, quercetin, rutin,
and luteolin [
101
–
104
] as well as some phenolic acids such as rosmarinic, sagerinic, syringic,
and salvianolic [105].
Among terpenoids, those of the di- type have been little studied regarding their
analgesic potential, however, an
in vitro
study of the anti-inflammatory effect of hardwickiic
acid is reported [
106
]. Finally, regarding triterpenoids, the anti-pain potential of oleanolic
and ursolic acids, as well as their derivatives, has been widely studied in nociception
models [
107
–
112
]. All of the above explains why, despite the different chemical profiles
of S. cinnabarina,S. lavanduloides, and S. longispicata, all three have antinociceptive effects,
since both shared and exclusive metabolites have shown good results in evaluations of
their analgesic properties.
The background on the analgesic effect of Salvia and the agreement with the results
obtained suggest a high potential as a medicinal alternative for pain relief, due to the
synergy of terpene and phenolic molecules present in the genus.
Due to the need to know the safety of the plants used in traditional Mexican medicine,
an evaluation of the acute toxicity of methanol extracts at a dose of 2000 mg/kg p.o. of S.
cinnabarina,S. lavanduloides, and S. longispicata was carried out, the results of which place
them at a non-toxic level of use (OECD 2001) [
44
]. The safety of use of these sages is consis-
tent with the acute toxicity results of other species of the genus, with an
LD50 > 2000 mg/kg,
p.o. calculated for extracts. For example, for the hydroalcoholic extract of S. officinalis leaves,
an LD
50
= 44.75 g/kg, p.o. was reported [
38
]. The infusion of S. circinata presented an
LD50 = 5 g/kg, p.o. [113]
and an LD
50
> 2000 mg/kg, administered intraplantarly (i. p.) [
34
].
Molecules 2024,29, 5465 11 of 18
While for S. hypoleuca Benth. the LD
50
= 1800 mg/kg, i. p. [
114
]. Finally, for the extracts of
different polarity from the aerial part of S. purpurea and S. semiatrata, the LD
50
was greater
than 2000 mg/kg, o.p. [
35
,
36
], making them absolutely safe and therefore, good prospects
for use as novel analgesics agents.
4. Materials and Methods
4.1. Drugs and Reagents
Diclofenac (DCF) and 37% formalin were purchased from Merck México (Naucalpan,
Mexico, Mexico). Tween 80 and saline solution (SS) were purchased from Sigma-Aldrich (St.
Louis, MO, USA). The solvent (methanol HPLC grade) used for extraction was purchased
from Tecsiquim, S.A. de C.V. (Mexico City, Mexico). The analysis phytochemicals (methanol,
leucine enkephalin, acetonitrile, water, and formic acid) were LC-MS grade and purchased
from Sigma-Aldrich.
4.2. Collection of Plant Material
One kilogram of the aerial part of each of the sages was collected in the surroundings
of Miahuatlán, Oaxaca, in June 2019 (Table 2). Fresh plant material was placed in a drying
chamber at 32
◦
C. Salvias were identified by Ph.D. Martha J. Martínez Gordillo. Voucher
specimens of these samples were deposited at the FCME Herbarium of the Faculty of
Sciences (FCME), the National Autonomous University of Mexico (UNAM).
Table 2. Altitude, geographical position and voucher number of Salvia species.
Species Elevation
(masl) Geographical Position Voucher Number
FCME
S. cinnabarina 2712
16
◦
06
′
42.6
′′
N, 96
◦
28
′
22.1
′′
W
184,736
S. lavanduloides 2712
16
◦
06
′
42.6
′′
N, 96
◦
28
′
22.1
′′
W
184,738
S. longispicata 2301
19
◦
14
′
31.2
′′
N, 94
◦
38
′
25.8
′′
W
184,737
4.3. Preparations of the Extracts
The plant material of each sage was dried at room temperature and finely ground
with a blender. Then, five grams of dried and ground plant material were weighed in
quadruplicate in Falcon
®
conical tubes (Corning Inc., Corning, NY, USA), 50 mL of HPLC
grade methanol was added and placed in an ultrasonic bath (Branson Bransonic
®
Bath 2800,
Emerson Electric Co., St. Louis, MO, USA) for 20 min at room temperature and ultrasonic
wave frequency of 40 kHz. It was then filtered (medium-pore filter paper) and evaporated
to dryness with a rotary evaporator at a temperature of 45
◦
C (RE100-Pro Digital Rotary
Evaporator, DLAB SCIENTIFIC Co., Shunyi, Beijing, China). The dried extracts were stored
in amber glass vials away from sunlight and moisture.
4.4. Chemical Profiling via Ultra Performance Liquid Chromatography Coupled Mass Spectrometry
(UPLC-ESI+/−-MS-QTOF)
Chemical profiling of methanol extracts was performed as was previously described
by Monribot-Villanueva et al. (2020) [
115
]. The analysis was carried out using an ultra-
high-resolution chromatographic system (ACQUITY UPLC I-Class System, Waters Co.,
Milford, MA, USA) coupled with a quadrupole time of flight (QTOF) high-resolution mass
spectrometer (SYNAPT G2-Si Mass Spectrometry, Waters Co., Milford, MA, USA) with
an electrospray ionisation source in positive and negative mode. An ACQUITY UPLC
BEH C18 column (Waters Co., Milford, MA, USA) was used, with column and sample
temperatures of 40
◦
and 15
◦
C, respectively. The flow rate was 0.3 mL/min and 5
µ
L of
extract was injected. The mobile phase consisted of a gradient of water and acetonitrile,
both with 0.1% formic acid. The gradient conditions were 0–20 min linear gradient 1
−
99%
B, then 20–24 min an isocratic step at 99% B, next 24–25 min a linear gradient 90–1% B, and
finally an isocratic step at 1% B for 5 min (total run time 30 min). The mass spectrometer
Molecules 2024,29, 5465 12 of 18
conditions were: Capillary, sampling cone and source offset voltages of 3000, 40, and
80 V, respectively. Source and desolvation temperatures of 120 and 20
◦
C, respectively.
Desolvation gas flow was set at 600 L/h and the nebulizer pressure of 6.5 Bar. The peptide
leucine-enkephalin was used as the lock mass (556.2771, [M + H]
+
; 554.2615, [M
−
H]
−
).
The mass acquisition method used was MSe in high-resolution mode (>29,000 m/zfor
leucine-enkephalin in both ionisation modes) using a mass range of 50–1200 Da and a scan
time of 0.5 s. The collision energies for Function 1 were 6 V and for Function 2 were a ramp
from 10 to 30 V. Spectrometric data were acquired and processed with MassLynx version
4.1 and MarkerLynx version 4.1 software (WatersTM Corporation, Milford, MA, USA).
4.5. Pharmacological Evaluations
4.5.1. Animals
The pharmacological evaluation was carried out in male CD-1 mice (25–30 g of body
weight). The mice were provided by the biotherium of the Faculty of Sciences. Mice
were placed in acrylic boxes with water and food ad libitum and kept at a controlled
temperature of 22
±
2
◦
C, standard humidity (50
±
5%) and with a 12 h light/dark cycle.
All experimental procedures were carried out in accordance with the Official Mexican
Standards, NOM-062-ZOO-1999 [
116
], and International Standards for the Care and Use of
Laboratory Animals in Research guidelines. The protocol was accepted by the Committee
on Academic Ethics and Scientific Responsibility (CEARC for its acronym in Spanish) of
the Faculty of Sciences, UNAM, under the folio PI_2021_08_02_Aguirre. Extracts and the
reference drug were suspended in 0.9% SS and Tween 80. All treatments were administered
orally (p.o.) in a volume of 10 mL/kg of mouse body weight and were prepared on the day
of the experiment.
4.5.2. Acute Toxicity
The toxicity of Salvia extracts was evaluated following OECD’s test No. 423 (2001).
The experimental groups of three mice were administered a maximum dose of 2000 mg/kg,
p.o. with the methanol extracts. The mice were observed for fourteen days to record signs
of toxicity such as weight loss, motor incoordination, ataxia, respiratory arrest, or death.
4.5.3. Formalin Test
The experiments were divided into groups of five animals, which received the fol-
lowing treatment: saline solution (SS), reference drug (DFC, 10 mg/kg), and the dose of
methanol extract (300 mg/kg, p.o.). Once the acute oral toxicity was evaluated, a wide
window of therapeutic activity was obtained in which no toxic effects were observed. This
allowed the choice of a single dose of 300 mg/kg, in accordance with the background
obtained by the working group, which demonstrates the significant effect in various pain
models of extracts of different polarity from various species of Salvia (23–25). After
30 min
of treatment administration, animals were injected subcutaneously on the intraplantar
surface of the right hind limb with 20
µ
L of 1% formalin to produce a licking behaviour.
Individually, the mice were placed inside a glass cylinder, surrounded by mirrors, to facili-
tate viewing of the behaviour from all angles by the evaluator. Then, the time spent licking
the limb administered with the nociceptive agent was measured for 1 min every 5 min
for a period of 30 min. Two phases were recorded in this test: neurogenic (0–10 min) and
inflammatory (10–30 min) phases. A significant decrease in either phase was interpreted as
demonstrative of an antinociceptive effect [117].
4.6. Statistical Analysis
Spectrometric data were acquired and processed with the MassLynx v. 4.1 and Mark-
erLynx v. 4.1 software from Waters (Milford, MA, USA). The intensity of each ion was
normalised and filtered relative to the total ion count to generate a data matrix. Such
matrix included m/zvalues, retention times and normalised peak areas. The mass spectra
of the chromatographic peaks were compared with public spectral databases of FooDB,
Molecules 2024,29, 5465 13 of 18
MassBank, LOTUS, Scopus and UNIIQUIM, using a maximum mass error of
±
5 ppm as
an accuracy criterium of chemical identity. The m/zdataset underwent pre-processing,
which involved centring on the mean and scaling using the Pareto principle. Subsequently,
for pattern recognition, a PCA and a heat map were applied using the MetaboAnalyst
v. 6.0 (Xia Lab, Montreal, QC, Canada) platform. Data from the antinociceptive activity
experiments were statistically analysed using Prism 8 software v. 8.4.3 (GraphPad Software
Inc., Boston, MA, USA) and ANOVA, followed by Dunnett’s post hoc test, to compare
treatments against the vehicle group. A value of p> 0.05 was considered significant.
5. Conclusions
The untargeted metabolomic analysis and the review previously carried out on the
chemical constituents of salvias allowed for the chemical differentiation of S. cinnabarina,S.
lavanduloides, and S. longispicata, of which 46 compounds were identified. In this study, ad-
vanced analytical and chemometric techniques were used to identify bioactive compounds
and distinctive chemical markers of three Mexican sages, making it one of the few studies
that have used the metabolomic technique in Mexico. Likewise, the importance of the
synergy of the constituents of terpene and phenolic nature was visualised, which plays an
important role in the efficacy and safety of the use of salvias as an alternative therapy in the
treatment of pain. The results of this study reinforce the richness of secondary metabolism
as well as the therapeutic properties of Mexican Salvia species used in Traditional Medicine.
It also provides evidence that S. cinnabarina,S. lavanduloides, and S. longispicata may be a
source of effective and safe compounds with analgesic potential. Future trials evaluating
extracts and isolated compounds from Salvia in various biological models are necessary
to propose and integrate new drugs into healthcare due to the growing interest in finding
alternative therapies.
Author Contributions: Conceptualisation, N.O.-M. and E.A.-H.; methodology, N.O.-M., J.L.M.-V.,
E.A.-H., J.A.G.-A., M.J.M.-G. and F.A.B.-P.; software, N.O.-M.; investigation, N.O.-M., E.A.-H., M.J.M.-
G. and F.A.B.-P.; writing—original draft preparation, N.O.-M.; writing—review and editing, N.O.-M.,
J.L.M.-V.; E.A.-H., J.A.G.-A., M.J.M.-G., F.A.B.-P. and M.S.-H. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by UNAM-PAPIIT research grant [IN221221; IN215925].
Institutional Review Board Statement: The pharmacological evaluation protocol was accepted by
Committee on Academic Ethics and Scientific Responsibility (CEARC) of the Faculty of Sciences,
UNAM (PI_2021_08_02_Aguirre).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: This paper is part of the requirements for obtaining a Doctoral degree at the
Postgraduate in Biological Sciences, UNAM of N.O.-M. The project also had the following CONAH-
CYT graduate scholarship number: 793655 and the fellowship grant to J.A.G.-A. (CVU-43029) for this
sabbatical leave. The authors thank Agustín Carmona Castro and Armando Rodríguez Velasco, for
their technical assistance.
Conflicts of Interest: The authors declare no conflicts of interest.
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