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García-Nava et al. / Botanical Sciences
ChemiCal Constituents of Salvia urica epling, and their antihyperglyCemiC
and antipropulsive effeCts
XitlaliCk garCía-nava1,2, miguel valdes3,4, fernando Calzada4, elihú Bautista2*,
omar Cortezano-arellano5, denisse de loera1, itzi fragoso-martínez6 and martha martínez-gordillo7
1 Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico.
2 CONAHCYT-División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A. C., San Luis Potosí,
Mexico.
3 Instituto Politécnico Nacional, Sección de Estudios de Posgrado e Investigación, Escuela Superior de Medicina, Mexico City, Mexico.
4 Unidad de Investigación Médica en Farmacología, UMAE Hospital de Especialidades, Centro Médico Nacional Siglo XXI, IMSS,
Mexico City, Mexico
5 Instituto de Ciencias Básicas, Universidad Veracruzana, Xalapa, Veracruz, Mexico.
6 Red de Biodiversidad y Sistemática, Instituto de Ecología, A.C., Xalapa, Veracruz, México.
7 Herbario de la Facultad de Ciencias (FCME), Universidad Nacional Autónoma de México, Mexico City, Mexico.
*Corresponding author: francisco.bautista@ipicyt.edu.mx
Botanical Sciences
DOI: 10.17129/botsci.3368
Received: June 22, 2023, Accepted: October 4, 2023
On line rst: November 27, 2023
Phytochemistry / Fitoquímica
This is an open access article distributed under the terms of the Creative Commons Attribution License CCBY-NC (4.0) international.
https://creativecommons.org/licenses/by-nc/4.0/
Abstract
Background: Salvia urica Epling is taxonomically and phylogenetically related to Salvia amarissima Ortega. The last species has pharmaco-
logical relevance by its contents of bioactive metabolites. Nowadays, Salvia urica has no reports about its chemical constituents and pharma-
cological activities.
Hypothesis: Does the close relationship between S. amarissima and S. urica led both species produce similar specialized metabolites? Does
Salvia urica display similar pharmacological effects as S. amarissima?
Studied species: Salvia urica Epling (Lamiaceae).
Study site and dates: The plant material was collected in Teopisca, Chiapas, Mexico, in December 2021.
Methods: Metabolites of the acetone extract from Salvia urica were identied by GC-MS and HPLC-PDA proling. In parallel, a phytochemi-
cal study was conducted, and the individual constituents puried, previously characterized by 1D NMR, were assayed on antihyperglycemic
effect in diabetic mice and a charcoal-gum arabic-induced hyperperistalsis model in rats.
Results: The volatile compounds identied by GC-MS were alkanes, aromatics and triterpenes. The principal constituents of the acetone extract
of Salvia urica were amarissinin A and 5,6-dihydroxy-7,3’,4’-trimethoxyavone, which were also quantied by HPLC-PDA. The extract and
both metabolites isolated showed an antihyperglycemic effect on streptozotocin-induced diabetic mice, suggesting a possible synergic effect.
In addition, the compound 5,6-dihydroxy-7,3’,4’-trimethoxyavone (IC50 = 0.79 mg/kg) showed a better antipropulsive effect than loperamide
(IC50 = 16.6 mg/kg).
Conclusions: The phytochemical composition of an acetone extract of Salvia urica was determined by rst time. The metabolites isolated from
this plant support the phylogenetic relationship of S. urica with Salvia amarissima, and they showed antipropulsive and antihyperglycemic effects.
Keywords: Specialized metabolites, terpenoids, Salvia genus, sage, antidiarrheal, antidiabetic, chromatographic methods, NMR analysis.
Resumen
Antecedentes: Salvia urica Epling está relacionada taxonómica y logenéticamente con Salvia amarissima Ortega. Ésta última tiene relevancia
farmacológica por su contenido de metabolitos secundarios. A la fecha, Salvia urica no cuenta con reportes de sus constituyentes químicos y de
sus actividades farmacológicas.
Hipótesis: ¿La relación cercana entre S. amarissima y S. urica permite que ambas especies produzcan metabolitos similares de tipo terpenoide?
¿Salvia urica produce un efecto farmacológico similar a S. amarissima?
Especies de estudio: Salvia urica Epling (Lamiaceae)
Sitio y años de estudio: El material vegetal fue colectado en Teopisca, Chiapas, México, en diciembre de 2021.
Métodos: Los constituyentes de las partes aéreas de Salvia urica, se identicaron por medio del perlamiento por GC-MS y HPLC-PDA. Para-
lelamente, se realizó un estudio toquímico, y los constituyentes individuales, caracterizados por RMN de 1D, se evaluaron en un ensayo de
actividad antihiperglucémica y en un modelo de hiperperistalsis.
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Resultados: Los componentes volátiles identicados por GC-MS son de tipo alcano, aromático y triterpenoide. Los principales constitu-
yentes no volátiles, son amarissinina A y 5,6-dihidroxi-7,3’,4’-trimetoxiavona. Ambos metabolitos se cuanticaron por HPLC-PDA. El
extracto de acetona y los dos metabolitos aislados mostraron efecto antihiperglucémico en ratones diabéticos, sugiriendo que hay un posible
efecto sinérgico. El compuesto 5,6-dihidroxi-7,3’,4’-trimetoxiavona (IC50 = 0.79 mg/kg) mostró un mejor efecto antipropulsivo que lopera-
mida (IC50 = 16.6 mg/kg).
Conclusiones: Se identicaron por primera vez los metabolitos mayoritarios de Salvia urica. Dos de estos metabolitos se aislaron previamente
de Salvia amarissima, apoyando su relación logénetica. Los dos compuestos mostraron efecto antipropulsivo y antihiperlgucémico.
Palabras clave: Metabolitos especializados, terpenoides, género Salvia, antidiarreico, antidiabético, métodos cromatográcos, análisis por
RMN
The Lamiaceae family contains 7,139 species (Harley et al. 2005), and the genus Salvia groups nearly
1,000 of them. From these species, 306 are distributed in Mexico. Salvia urica Epling is a perennial
herbaceous plant native to Mexico, with a restricted distribution in the state of Chiapas, as well as in
Guatemala and Belize (Martínez-Gordillo et al. 2017). It grows at elevations from 400 to 2,600 meters
above mean sea level, in seasons ranging from summer to winter (Clebsch 2003). This plant is commonly known as
“pendolita morada”, “canastillas”, “tutzunún”, “Salvia bretónica”, and “chichingua azul” (Chicago Natural History
Museum 1973, Padilla-Gómez 2007). Some traditional medicinal uses given to S. urica are to treat “bilis”, diabetes
mellitus, stomachache, and diarrhea (Padilla-Gómez 2007). However, no phytochemical and pharmacological re-
ports of the constituents derived from this plant species support its ethnomedical uses in Mexico. To date, there is just
a report describing the antibacterial effect of a methanolic extract of the plant against Escherichia coli (Sakagami et
al. 2001). Originally, Salvia urica and Salvia amarissima Ortega were classied together in section Uricae Epling,
due to their morphological resemblance (Epling 1939). Later, they were moved to a broader section: Scorodonia
Epling (Epling 1941). Phylogenetic studies of Salvia subgenus Calosphace have shown that both these sections are
articial groupings (Fragoso-Martínez et al. 2018). However, Salvia amarissima and S. urica are closely related,
forming a clade with Salvia leucochlamys Epling, Salvia ozolotepecensis J.G.González & Fragoso, Salvia perlonga
Fernald, and Salvia praestans Epling (González-Gallegos et al. 2019). The lack of phytochemical and pharmacologi-
cal studies, and the phylogenetic closeness between S. urica and S. amarissima prompted us to study the rst species;
in order to explore if it produces secondary metabolites of diterpenoid-type that supports this relationship since a
chemotaxonomic approach, and if it shares pharmacological properties with S. amarissima.
As part of our ongoing efforts to study the phytochemical composition of Mexican medicinal plants for drug
discovery and agrochemical purposes (Ortega et al. 2020, Bautista et al. 2022, García-Nava et al. 2022), herein, we
describe the isolation and identication of the major metabolites from an acetone extract of Salvia urica, as well as
the subsequent determination of the anti-hyperglycemic and anti-propulsive effects of these constituents.
Materials and methods
General experimental procedures. 1D NMR experiments were performed on an Agilent Mercury NMR spectrometer
500 MHz, equipped with a gradient probe for variable temperature experiments. Chemical shifts were referred to
TMS, and J values are given in Hertz (Hz). Column chromatography (CC) was performed on silica gel 60 (Merck G).
Thin-Layer Chromatography (TLC) was carried out on precoated Macherey-Nagel Sil G/UV254 plates of 0.25 mm
thickness, and spots were visualized by spraying with 3 % Ce(SO4)2 in H2SO4 2 N, followed by heating. The GC-MS
analyses were obtained on an Agilent gas chromatograph, model GC-7890b coupled to an Agilent mass spectrometer
EM-5877A, using a GC capillary column DB 5ht (30 m × 0.320 mm i.d. × 0.1 μm, Agilent). The HPLC analyses were
obtained on a 1200 HPLC Agilent chromatograph equipped with a G1315D Agilent PDA detector and a G1315A Agi-
lent uorescence detector. HPLC separation was performed in a Synergi Polar RP column (Phenomenex, 5 mm, 4.6 ×
250 mm) with a gradient of acetic acid 1 % (A) and acetonitrile (B) at the following times: 0 min, 80 % A; 1 min, 80 %
A; 31 min, 40 % A, 33 min, 20 % A; 43 min, 20 % A. The chromatograms were obtained at 30 °C; the standards used
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were amarissinin A (1) (> 98 %, HPLC-PDA) and 5,6-dihydroxy-7,3’,4’-trimethoxyavone (2) (> 98 %, HPLC-PDA);
and the calibration curves of the standards were made using different concentrations 0.25-0.00625 mg/mL.
Plant material. The aerial parts were collected at the owering stage in Teopisca, Chiapas, Mexico, in December
2021. A voucher specimen was deposited at the Herbarium of Facultad de Ciencias, UNAM (FCME-180582), and
determined by Dr. Martha Martínez Gordillo.
Extraction and isolation. The dried and grounded aerial parts of Salvia urica (218 g) were subjected to extraction
by maceration with acetone (3 × 2 L). The extract was ltered, and the solvent was recovered by distillation at re-
duced pressure to obtain 11 g of dried residue. The S. urica acetone extract (SUE) was submitted to silica gel CC
(8.5 × 4.5 cm, 125 mL), eluting with hexane-EtOAc, EtOAc, and EtOAc-MeOH mixtures in increasing polarity. The
grouping fractions was conducted by their likeness observed in the TLC analyses as follows: Fraction A (frs.1-7,
eluted with hexanes-EtOAc 9:1), fraction B (frs. 8-22, eluted with hexanes-EtOAc 8:2), fraction C (frs. 23-27, eluted
with hexanes-EtOAc 3:2), fraction D (frs. 28-38, eluted with hexanes-EtOAc 3:2), fraction E (frs. 39-43, eluted with
hexanes- EtOAc 1:1), fraction F (frs. 44-52, eluted with hexanes-EtOAc 3:7), fraction G (fr. 53, eluted with EtOAc),
fraction H (fr. 54, eluted with EtOAc), fraction I (frs. 55-58, eluted with EtOAc-MeOH 9:1). Fraction D (1.6 g) was
submitted to silica gel CC (10.0 × 2.0 cm, 25 mL), eluting with CHCl3-MeOH mixtures in increasing polarity to give
ve fractions (D1-D5). Fr. D2 (eluted with CHCl3-MeOH 97:3) yielded 62 mg of amarissinin A (1) by sequential
crystallizations from acetone/hexanes, EtOAc/hexanes, and acetone/EtOAc. Fr. E yielded 7 mg of 5,6-dihydroxy-
7,3’,4’-trimethoxyavone (2), which was ltrated, and crystallized from EtOAc/hexanes.
Compound 1.- yellowish crystals, mp 219-224 °C; 1H NMR (500 MHz, CDCl3-DMSO-d6, Figures S1 and S2): δH
7.82 (br s, H-16), 7.39 (t, J = 1.8 Hz, H-15), 6.69 (ddd, J = 10.1, 4.9, 2.2 Hz, H-2), 6.54 (br d, J = 1.8 Hz, H-14), 6.12
(br s, H-11), 5.92 (dt, J = 10.1, 1.4 Hz, H-1), 4.72 (dd, J = 8.7, 1.5 Hz, H-19Pro R), 4.35 (d, J = 8.7 Hz, H-19Pro S), 2.71
(dd, J = 19.3, 4.9 Hz, H-3a), 2.61 (dt, J = 19.3, 2.2 Hz, H-3b), 2.47 (ddd, J = 13.1, 12.0, 5.1 Hz, H-7a), 2.21 (ddd, J
= 13.5, 11.6, 5.4 Hz, H-7b), 2.03 (s, H3-20), 1.61 (m, H-6a), 1.56 (m, H-6b); 13C NMR (125 MHz, DMSO-d6, Figure
S3): δC 197.39 (C, C-10), 173.89 (C, C-18), 161.75 (C, C-17), 151.47 (C, C-12), 150.37 (C, C-9), 143.96 (CH, C-15),
143.04 (CH, C-2), 141.48 (CH, C-16), 127.39 (CH, C-1), 120.72 (C, C-8), 119.31 (C, C-13), 106.62 (CH, C-14),
104.60 (CH, C-11), 75.81 (C, C-4), 69.49 (CH2, C-19), 57.18 (C, C-5), 29.84 (CH2, C-3), 28.79 (CH2, C-6), 21.95
(CH2, C-7) and 18.55(CH3, C-20).
Compound 2.- yellowish crystals, mp 240-241 °C; 1H NMR (500 MHz, DMSO-d6, Figure S4): δH 12.62 (s, OH-5),
8.72 (s, OH-6), 7.70 (d, J = 8.5 Hz, H-5’), 7.59 (br s, H-2’), 7.13 (dd, J = 8.5, 1.6 Hz, H-6’), 6.98 (s, H-8), 6.94 (s,
H-3), 3.88 (OCH3-7), 3.85 (OCH3-3’), 3.80 (OCH3-4’); 13C NMR (125 MHz, DMSO-d6, Figure S5): δC 182.26 (C,
C-4), 163.34 (C, C-2), 154.41 (C, C-7), 152.04 (C, C-4’), 149.03 (C, C-3’), 146.16 (C, C-5), 129.98 (C, C-6), 123.07
(C, C-1’), 119.98 (CH, C-6’), 111.7 (CH, C-5’), 109.39 (CH, C-2’), 103.45 (CH, C-3), 91.26 (CH, C-8), 56.16 (CH3,
OCH3-7), 55.73 (CH3, OCH3-3’), 55.58 (CH3, OCH3-4’).
Identication of the volatile constituents by GC-MS. A sample of 5 g of plant material was macerated with acetone
(3 × 60 mL) for 48 h to obtain residues ranging from 490 to 503 ± 9.9 mg (5.5 ± 0.08 % yield dry weight). Then, 10 mg
of extract were dissolved in 2 mL of acetone using sonication, and a volume of 0.2 μL containing this solution was
injected into the gas chromatograph (splitless). The temperature of the port injection was maintained at 220 °C, and
the oven was 80 °C for 2 min, hereafter increased at 3, 5, and 10 °C/min until 100, 150, and 330 °C, respectively.
The nal temperature was maintained for 9 min. Each analysis was repeated in triplicate, and the volatile com-
pounds were identied by deconvolution, using the W10N11 database (Wiley10Nist11) and the % area normalized
(Sepúlveda-Cuellar et al. 2021).
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High Performance Liquid Chromatography. A sample of plant material (10 g) was macerated with acetone (3 × 60 mL)
for 48 h. This procedure was repeated with three independent samples to obtain residues between 490–503 ± 9.9 mg.
Then, a solution of each extract at 2.5 mg/mL was prepared in methanol. The injection volume of standards was 5 μL,
and 10 μL for samples, using a ow rate of 0.8 mL/min. The temperature of the equipment was 30 °C. The PDA detector
acquires the chromatograms at 240 and 330 nm, and the FL detector operates with an excitation longwave of 250 nm, and
an emission longwave of 410 nm. All experiments were repeated in triplicate according to Sepúlveda-Cuellar et al. (2021).
Animals. Balb/c male mice of 8-10 weeks of age weighing 25 ± 5 g, and with glucose level values of 161 ± 5 mg/mL
were used for the antihyperglycemic test. Sprague-Dawley male rats (200-250 g) for the antipropulsive test were
used. The animals were obtained and raised in the Animal House of the National Medical Center “Siglo XXI” at In-
stituto Mexicano del Seguro Social (IMSS). The in vivo experiments were conducted following the Ofcial Mexican
NOM-0062-ZOO-1999 (SEMARNAT 1999) for Animal Experimentation and Care. The room temperature was 22 ±
2 °C with a 12-h light–dark natural cycle to maintain the animal care. The animals were fed with a standard diet and
water ad libitum. All in vivo experiments were conducted with the approval of the Specialty Hospital Ethical Com-
mittee of the National Medical Center “Siglo XXI” at IMSS (register: R-2019-3601-004).
Induction of experimental type 2 diabetes in mice. The diabetes was induced in male Balb/c mice using the strepto-
zotocin/nicotinamide model, according to a minor modication of the method described by Valdés et al. 2019. The
mice fasted for 16 h before being treated with an intraperitoneal solution of streptozotocin (100 mg/kg). After 30 min
of the streptozotocin administration, the mice were treated with nicotinamide (240 mg/kg in cold saline solution) via
intraperitoneal. The mice were fed with sucrose solution (10 %) ad libitum over three days at the end of the third day.
The sucrose solution was retired and substituted with water on the fth day. After 24 h, blood glucose levels were
measured by the glucose oxidase method (ACCU-CHECK® Instant Blood Glucose System, Roche, DC®, Mexico).
Antihyperglycemic effect. Balb/c mice were randomly divided into 8 groups (n = 6 per group). The groups were
separated by treatment as follows: Normoglycemic (NM control) and Diabetic (SID2 control) mice treated with
vehicle (2 % Tween 20 in water); NM mice and SID2 treated with SUE (300 mg/kg), compound 1 (50 mg/kg) and
compound 2 (50 mg/kg), respectively. The collection of blood samples from the tail vein was at 0, 2, and 4 h. The
analysis of blood samples was done by the glucose oxidase method (Valdés et al. 2019). The results were expressed
as mean values ± standard error of the mean (SEM). The statistical analyses were performed by GraphPad Prism
(GraphPad Software Inc., San Diego, CA, USA). The statistical evaluation was conducted by Bonferroni test for
multiple comparisons with a P < 0.05 of signicance.
Antipropulsive effect. The rats were fasted for 12 h before starting the experiment, but with water access ad libitum.
The method reported by Calzada et al. (2010) was followed with minor modications. The groups were divided into
control and test groups (n = 6 per group). The control group was treated with vehicle (1 mL 2 % DMSO in water) or
loperamide hydrochloride (10 mg/kg, 1 mL in 2 % DMSO, positive control), and the test groups were divided into
SUE (12.5 - 50 mg/kg), compound 1 (0.125 - 1.5 mg/kg) and compound 2 (0.125 - 1.5 mg/kg). After 20 min, the ani-
mals were administered with 1 mL of charcoal meal [10 % charcoal suspension in 5 % aqueous gum Arabic] by oral
route. After 30 min, the animals were sacriced, and their stomach and small intestine were removed and extended on
a glass surface. The distance from the pylorus to the caecum was measured and expressed as a percentage. All results
were expressed as mean ± S.E.M. and evaluated by Student’s t-test with a P < 0.05 of signicance.
Results
The GC-MS analysis of the volatile constituents from the acetone extract of Salvia urica indicated the presence of
16 metabolites, which include six triterpenes, seven alkanes and three aromatic metabolites such as anthraquinone
and naphthol derivatives (Supplementary material, Table S1). The relative amounts of the volatile compounds, by
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class, were: 76.61, 15.39, and 7.99 %, respectively. The HPLC-PDA proling of the non-volatile constituents in the
acetone extract of S. urica, followed by the subsequent quantication of the isolated compounds (Supplementary
material, Figures S1-S5) by chromatographic methods, indicated that amarissinin A (1, rt = 24.24 min, 330 nm) and
5,6-dihydroxy-7,3’,4’-trimethoxyavone (2, rt = 24.58 min, 330 nm) are the major constituents in the extract (Figure
2, Table 1).
Figure 2. (A) HPLC-PDA proling chromatogram of amarissinin A (1). (B) HPLC-PDA proling chromatogram of 5,6-dihydroxy-7,3’,4’-trimethoxy-
avone (2). (C) HPLC-PDA proling chromatogram of the acetone extract of Salvia urica at 330 nm.
Figure 1. Chemical structures of amarissinin A (1) and 5,6-dihydroxy-7,3’,4’-trimethoxyavone (2).
In the normoglycemic mice, the extract and the metabolites isolated from Salvia urica did not signicantly affect
glucose levels (Table 2). However, in streptozotocin-induced type 2 diabetes mice, the extract and the metabolites
showed an antihyperglycemic effect at 2 and 4 h after their administration (Table 2). Compound 2 showed a dose-
dependent decrement in glucose levels; and, the acetone extract showed the lowest glucose level after 4 h of treat-
ment. A synergic effect between the constituents of the extract is possible.
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On the charcoal-gum Arabic-induced hyperperistalsis model in rats, the extract, and their major metabolites of
Salvia urica were tested at different doses. All showed a dose-dependence effect. Among them, amarissinin A (1)
showed the best inhibitory effect on the peristalsis at 0.125 mg/kg, and the 5,6-dihydroxy-7,3’,4’-trimethoxyavone
(2) showed a half maximal effective concentration of 0.79 mg/kg, in comparison with the positive control (1.003 mg/
kg, Table 3).
Acetone extract (yield in % w/w) Compound 1 (mg/g extract) Compound 2 (mg/g extract)
5.17 ± 0.09 163.36 ± 3.17 141.98 ± 4.59
Table 1. Quantication of amarissinin A (1) and 5,6-dihydroxy-7,3’,4’-trimethoxyavone (2) by HPLC-DAD (at 330 nm) in the total
extract from Salvia urica.
Note: Data expressed as average ± SD.
Treatment
Blood glucose levels (mg/dL)
0h 2h 4h
NM control 137.5 ± 5.6 131.3 ± 3 133 ± 3.5
SID2 control 300 ± 32.7 356.3 ± 21.5 351.3 ± 25.2
SID2 + SUE (300 mg/kg) 308.2 ± 7.6 285.2 ± 19 173 ± 31.7*, b
SID2 + 1 (50 mg/kg) 297.3 ± 6.9 337.8 ± 32.6 302.3 ± 45.4
SID2 + 2 (50 mg/kg) 289.8 ± 8.5 252.3 ± 5.4*, a237.3 ± 5.6*, b
NM + SUE (300 mg/kg) 134.5 ± 3.7 128.3 ± 5.2 134.5 ± 3.1
NM + 1 (50 mg/kg) 136 ± 4.9 136.5 ± 5.3 133.8 ± 5
NM + 2 (50 mg/kg) 134.8 ± 2.5 126.8 ± 2.2 130.3 ± 2
Table 2. Blood glucose levels of male normoglycemic mice (NM) and streptozotocin-induced type 2 diabetes mice (SID2) at 0, 2 and
4 h, on the acute antihyperglycemic test.
Discussion
The species of the Salvia genus have broad traditional medicinal uses in Mexico, which include treating digestive and
gynecological problems, and affections of the nervous system (Jenks & Kim 2013). Previous reports have explored
the phylogenetic relationships of species from Salvia subgenus Calosphace based on nuclear and plastid markers
(ITS, trnL-trnF, and trnH-psbA) (Fragoso-Martínez et al., 2018; González-Gallegos et al. 2018), and nuclear loci
from massive sequencing data (Lara-Cabrera et al. 2021). Using the phylogeny of Salvia subgenus Calosphace as a
tool to target specic metabolites as proposed by Ortiz-Mendoza et al. (2022), we decided explore the phytochem-
istry of key species; since we analyzed a species that was closely related to S. amarissima, which belongs to a taxon
that has shown a wide variety of metabolites, which include neo-clerodanes, 9,10-seco-neo-clerodanes, and amaris-
sanes (Bautista et al. 2016). The major metabolites found in S. urica: amarissinin A (1) and 5,6-dihydroxy-7,3’,4’-
trimethoxyavone (2), also are produced in S. amarissima, supporting their phylogenetic closeness. In addition, the
occurrence of compounds 1 and 2 supports the hypothesis that S. urica has similar pharmacological effects like to its
congener. A previous study analyzed the inhibitory effect of methanolic extracts of plants on the production of vero-
The data are expressed as mean ± S.E.M, n = 6; *P < 0.05 compared to the initial value; aP <0.05 compared
to DM2 control 2h, b P <0.05 compared to DM2 control 4h. SID2: Streptozotocin-induced type 2 diabetic
mice, SUE: Acetone extract of Salvia urica; 1: Amarissinin A; 2: 5,6-dihydroxy-7,3’,4’-trimethoxyavone.
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toxin by the enterohemorrhagic strain of Escherichia coli O157:H7 (VEc) (Sakagami et al. 2001). In that study, the
extract from S. urica showed an inhibitory effect on VEc. Likewise, Calzada et al. (2020) reported that compounds 1
and 2, isolated from S. amarissima, showed antiprotozoal activity against Entamoeba histolytica and Giardia lamblia
with IC50 values ranging from moderate to good activity (62.1-101.1 and 0.05-0.13 μM, respectively), compared with
positive control (0.23-1.22 μM, metronidazole). In the present study, the compounds 1 and 2, but now isolated from
S. urica, showed an antipropulsive effect on charcoal-gum Arabic-induced hyperperistalsis model in rats, providing
new insights to understand underlying ways to stop diarrhea; and provided evidence-based support for the traditional
uses of both plant species. Concerning to antidiabetic effect, previous reports have shown that the antihyperglycemic
activity of a total extract from S. amarissima is associated with an α-glucosidase inhibitory activity. The subsequent
phytochemical analysis of the extract led to the isolation of compound 2, as the more active constituent (IC50 1 800
μM) of the extract. This activity was even higher than those for diterpenes (IC50 > 10 000 μM) also isolated, and close
near to positive control (IC50 100 ± 0.3 μM, acarbose) (Flores-Bocanegra et al. 2017, Solares-Pascacio et al. 2021).
Considering the above, it is plausible that the presence of compound 2 contribute signicantly to the antihyperglyce-
Treatment Hyperperistalsis
(cm)
Antipropulsive
Effect (%)
CE50
(mg/kg)
2 h
AC 75.3 ± 2 - -
AC-GA 91.6 ± 2.7 - -
AC-GA + SUE 50 mg/kg 78 ± 1 83.6 ± 6.6 16.65
25 mg/kg 80.3 ± 1 69.3 ± 6.2
12.5 mg/kg 85.3 ± 0.6 38.7 ± 3.8
AC-GA + 1 1.5 mg/kg 79.3 ± 1.6 75.5 ± 10.4 0.81
0.5 mg/kg 83.6 ± 1.6 48.9 ± 10.1
0.25 mg/kg 86 ± 1.4 34.6 ± 9
0.125 mg/kg 91 ± 0.4 4 ± 2.4
AC-GA + 2 1.5 mg/kg 63.1 ± 1.19 100 0.79
0.5 mg/kg 86.3 ± 0.2 32.6 ± 1.4
0.25 mg/kg 90 ± 0.4 10.2 ± 2.4
0.125 mg/kg 97.3 ± 1.3 0
AC-GA + C 5 mg/kg 39.6 ± 6.5 100 1.003
2.5 mg/kg 77.6 ± 6.1 85.7 ± 5.2
1.25 mg/kg 83 ± 0.4 46.9 ± 2.4
Table 3. Antipropulsive effect Salvia urica and loperamide products on charcoal activated-gum Arabic induced hyperperistalsis
model in rat.
Antipropulsive effect calculated after administration of the treatments. Values expressed as means ± SEM,
n = 6, CE50: half maximal effective concentration. AC: Activated charcoal; GA: Gum Arabic; SUE: Ac-
etone extract of Salvia urica; 1: Amarissinin A; 2: 5,6-dihydroxy-7,3’,4’-trimethoxyavone; C: Loperamide
chloride.
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Chemical constituents of Salvia urica
8
mic effect of S. urica, through the inhibition of α-glucosidase. In addition, it is worthy to note that was not detected the
presence of oleanolic and ursolic acids, and stigmasterol in the extract of S. urica; which in turn, have been identied
as the bioactive constituents, responsible for the antihyperglycemic properties of Salvia species (Santos et al. 2012,
Bakrim et al. 2022, Ortega et al. 2022). The triterpenoids α- and β-amyrins were also detected in signicant amounts
in the acetone-soluble extract from S. urica by GC-MS (Supplementary material, Table S1). The relative amount (%)
of each triterpenoid in the volatile portion of the extract was: 17.38 ± 8.70 and 25.84 ± 0.98, respectively. Both com-
pounds have been reported as α-glucosidase inhibitors, and are involved in the improving of the insulin plasm levels
and exert a protective effect of β-cells; and prevent the resistance to insulin via AMPK signaling induction (Mabhida
et al. 2018, Dirir et al. 2022, Entezari et al. 2022). Due the above, amyrins are considered as multi-target compounds,
therefore additional studies with the acetone extract of S. urica and their individual constituents are necessary to ex-
plore other action mechanisms that led to understand the antihyperglycemic effect of the plant.
In conclusion, the phytochemical study of an acetone-soluble extract from the aerial parts of S. urica led to the
identication of its major constituents, their quantication through HPLC-PDA, and the evaluation of their antihy-
perglycemic and antiperistaltic effects, providing evidence-based support for the traditional medicinal uses of the
plant. Finally, the pharmacological effect displayed by the diterpenes derived from Salvia amarissima as multidrug-
resistance modulators in cancer cells (Bautista et al. 2016), could prompt exploring these properties in Salvia urica.
Acknowledgments
The authors thank to Dr. Gerardo A. Salazar IB-UNAM, for the photo of Salvia urica kindly provided.
Declaration of competing interests
The authors declare that there is no conict of interest, nancial or personal, in the information, presentation of data
and results of this article.
Supplementary material
Supplemental material for this article can be accessed here https://doi.org/10.17129/botsci.3368
Literature cited
Bakrim S, Benkhaira N, Bourais I, Benali T, Lee LH, Omari NE, Sheikh RA, Goh KW, Ming LC, Bouyahya A.
2022. Health Benets and Pharmacological Properties of Stigmasterol. Antioxidants 11 : 1912. DOI: https://doi.
org/10.3390/antiox11101912
Bautista E, Fragoso-Serrano M, Ortiz-Pastrana N, Toscano RA, Ortega A. 2016. Structural elucidation and evalua-
tion of multidrug-resistance modulatory capability of amarissinins A–C, diterpenes derived from Salvia amaris-
sima. Fitoterapia 114: 1-6. DOI: https://doi.org/10.1016/j.tote.2016.08.007
Bautista E, Lozano-Gamboa S, Fragoso-Serrano M, Rivera-Chávez J, Salazar-Olivo LA. 2022. Jatrophenediol, a
pseudoguaiane sesquiterpenoid from Jatropha dioica rhizomes. Tetrahedron Letters 104: 154040. DOI: https://
doi.org/10.1016/j.tetlet.2022.154040
Calzada F, Arista R, Pérez H. 2010. Effect of plants used in Mexico to treat gastrointestinal disorders on char-
coal-gum acacia-induced hyperperistalsis in rats. Journal of Ethnopharmacology 128: 49-51. DOI: https://doi.
org/10.1016/j.jep.2009.12.022
Calzada F, Bautista E, Barbosa E, Salazar-Olivo LA, Alvidrez-Armendáriz E, Yepez-Mulia L. 2020. Antiprotozoal
activity of secondary metabolites from Salvia circinata. Revista Brasileira de Farmacognosia 30: 593-596. DOI:
https://doi.org/10.1007/s43450-020-00077-7
Botanical Sciences / Online First
Botanical Sciences / Online First
9
García-Nava et al. / Botanical Sciences
Chicago Natural History Museum. 1973. Fieldiana Botany. In: Chicago Natural History Museum 24: 300. USA.
Clebsch B. 2003. The new book of Salvias: Sages for every garden. Portland, USA: Timber Press. ISBN-13: 978-0-
88192-913-3
Dirir AM, Daou M, Yousef AF, Yousef LF. 2022. A review of alpha-glucosidase inhibitors from plants as poten-
tial candidates for the treatment of type-2 diabetes. Phytochemistry Reviews 21: 1049-1079. DOI: https://doi.
org/10.1007/s11101-021-09773-1
Entezari M, Hashemi D, Taheriazam A, Zabolian A, Mohammadi S, Fakhri F, Hashemi M, Hushmandi K, Ashraza-
deh M, Zarrabi A, Nuri-Ertas Y, Mirzaei S, Samarghandian S. 2022. Biomedicine & Pharmacotherapy 146:
112563. DOI: https://doi.org/10.1016/j.biopha.2021.112563
Epling C. 1939 A revision of Salvia subgenus Calosphace. Repertorium Specierum Novarum Regni Vegetabilis.
Dahlem, Berlin: Repertoriums.
Epling C. 1941 Supplementary notes on American Labiatae. II. Bulletin Torrey Botanical Club 68:552-568.
Flores-Bocanegra L, González-Andrade M, Bye R, Linares E, Mata R. 2017. α-Glucosidase Inhibitors from Salvia
circinata. Journal of Natural Products 80: 1584-1593. DOI: https://doi.org/10.1021/acs.jnatprod.7b00155
Fragoso-Martínez I, Martínez-Gordillo M, Salazar GA, Sazatornil F, Jenks AA, García-Peña MR, Barrera-Aveleida
G, Benítez-Vieyra S, Magallón S, Cornejo-Tenorio G, Granados-Mendoza C. 2018. Phylogeny of the Neotropical
sages (Salvia subg. Calosphace; Lamiaceae) and insights into pollinator and area shifts. Plant Systematics and
Evolution 304: 43-55, https://doi.org/10.1007/s00606-017-1445-4
García-Nava X, Fragoso-Serrano M, de Loera D, Cortezano-Arellano O, Calzada F, Bedolla-García BY. 2022. Ama-
risolide H and 15-epi-Amarisolide H, two diterpenoid glucosides from Salvia circinnata. Revista Brasileira de
Farmacognosia 32: 993-999. DOI: https://doi.org/10.1007/s43450-022-00332-z
González-Gallegos JG, Fragoso-Martínez I, González-Adame G, Martínez-Ambriz AE, López-Enríquez IL. 2018.
Salvia ozolotepecensis, S. patriciae and S. sirenis (Lamiaceae), three new species from Miahuatlán district,
Oaxaca, Mexico. Phytotaxa 362: 143-159. DOI: https://doi.org/10.11646/phytotaxa.362.2.2
Harley RM, Atkins S, Budantsev AL, Cantino PD, Conn BJ, Grayer R, Harley MM, de Kok R, Krestovskaja T, Mo-
rales R, Paton AJ, Ryding O, Upson T. 2005. The Families and Genera of Vascular Plants. Taxon, 54: 574. DOI:
https://doi.org/10.2307/25065407
Jenks A, Kim SC. 2013. Medicinal plant complexes of Salvia subgenus Calosphace: An ethnobotanical study of new
world sages. Journal of Ethnopharmacology 146: 214-224. DOI: https://doi.org/10.1016/j.jep.2012.12.035
Lara-Cabrera SI, Perez-Garcia MdlL, Maya-Lastra CA, Montero-Castro JC, Godden GT, Cibrian-Jaramillo A, Fisher
AE, Porter JM. 2021. Phylogenomics of Salvia L. subgenus Calosphace (Lamiaceae). Frontiers in Plant Science
12: 725900. DOI: https://doi.org/10.3389/fpls.2021.725900
Mabhida SE, Dludla PV, Johnson R, Ndlovu M, Louw J, Opoku AR, Mosa RA. 2018. Protective effect of triterpenes
against diabetes-induced β-cell damage: An overview of in vitro and in vivo studies. Pharmacological Research
137: 179-192. DOI: https://doi.org/10.1016/j.phrs.2018.10.004
Martínez-Gordillo M, Bedolla-García B, Cornejo-Tenorio G, Fragoso-Martínez I, García-Peña MdR, González-
Gallegos JG, Lara-Cabrera SI, Zamudio S. 2017. Lamiaceae de México. Botanical Sciences 95:780-806. DOI:
https://doi.org/10.17129/botsci.1871
Ortega A, Pastor-Palacios G, Ortiz-Pastrana N, Ávila-Cabezas E, Toscano RA, Joseph-Nathan P, Morales-Jiménez J,
Bautista E. 2020. Further galphimines from a new population of Galphimia glauca. Phytochemistry 169: 112180.
DOI: https://doi.org/10.1016/j.phytochem.2019.112180
Ortega R, Valdés M, Alarcón-Aguilar FJ, Fortis-Barrera A, Barbosa E, Velázquez C, Calzada F. 2022. Antihypergly-
cemic Effects of Salvia polystachya Cav. and Its Terpenoids: Glucosidase and SGLT1 Inhibitors. Plants 11: 575.
https://doi.org/10.3390/plants11050575
Ortiz-Mendoza N, Aguirre-Hernández E, Fragoso-Martínez I, González-Trujano ME, Basurto-Peña FA, Martínez-
Gordillo MJ. 2022. A review on the ethnopharmacology and phytochemistry of the Neotropical sages (Salvia
Botanical Sciences / Online First
Botanical Sciences / Online First
Chemical constituents of Salvia urica
10
subgenus Calosphace; Lamiaceae) emphasizing Mexican species. Frontiers in Pharmacology 13: 867-892. DOI:
https://doi.org/10.3389/fphar.2022.867892
Padilla-Gómez E. 2007. Estudio ecológico y etnobotánico de la vegetación del Municipio de San Pablo Etla, Oaxa-
ca. Ms Thesis. Instituto Politécnico Nacional. α
Sakagami Y, Murata H, Nakanishi T, Inatomi Y, Watabe K, Iinuma M, Tanaka T, Murata J, Lang FA. 2001. Inhibitory
effect of plant extracts on production of Verotoxin by enterohemorrhagic Escherichia coli O157:H7. Journal of
Health Sciencie 47: 437-477. DOI: https://doi.org/10.1248/jhs.47.473
Santos FA, Frota JT, Arruda BR, de Melo TS, da Silva AA, Brito GAC, Chaves MH, Rao VS. 2012. Antihyperglyce-
mic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice. Lipids
in Health and Disease 11: 98. DOI: https://doi.org/10.1186/1476-511X-11-98
SEMARNAT. [secretaria del Medio Ambiente y Recursos Naturales]. 1999. NOM-062-ZOO-1999: Especicaciones
técnicas para la producción, cuidado y uso de los animales de laboratorio. Ciudad de México: Diario Ocial de la
Federación (miércoles 22 de agosto de 2001).
Sepúlveda-Cuellar L, Duque-Ortiz A, Yáñez-Espinosa L, Calzada F, Bautista E, Pastor-Palacios G, Bedolla García
BY, Flores-Rivera J, Badano EI, Douterlungne D. 2021. Phylogenetic and Chemical Analyses of the Medicinal
Plant Salvia circinnata: an Approach to Understand Metabolomics Differences. Revista Brasileira de Farmacog-
nosia 31: 676-688. DOI: https://doi.org/10.1007/s43450-021-00168-z
Solares-Pascacio JI, Ceballos G, Calzada F, Barbosa E, Velazquez C. 2021. Antihyperglycemic and Lipid Prole
Effects of Salvia amarissima Ortega on Streptozocin-Induced Type 2 Diabetic Mice. Molecules 26: 947. DOI:
https://doi.org/10.3390/molecules26040947
Valdés M, Calzada F, Mendieta-Wejebe JE. 2019. Structure-activity relationship study of acyclic terpenes in blood
glucose levels: potential α-glucosidase and sodium glucose cotransporter (SGLT-1) inhibitors. Molecules 24:
4020. DOI: https://doi.org/10.3390/molecules24224020
Associate editor: Juan Rodrígo Salazar
Author contributions: EB designed the project, provided nancial support, and carried out the phytochemical study. XGN carried out the
phytochemical study. FC and MV conducted the biological assays. OCA and DL acquired and analyzed spectroscopic and spectrometric data.
IFM and MMG collected and identied the plant material studied. All the authors contributed to the writing of the manuscript and approved the
nal version.
Supporting agencies: Consejo Nacional de Ciencia y Tecnologia (CONACYT, project CB-A1-S-7705). X. Garcia (CVU 740455) and E. Bau-
tista are grateful to CONACYT for the Research Fellowships.
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