Plant and Growth Condition Interactions on the Phenolic Compound Contents and Antioxidant Activity in Salvia circinata Cav., a Medicinal Sage

Abstract

The genus Salvia has a worldwide distribution, and its contribution to traditional medicine and as an aromatic plant has been recognized since ancient times, with few documented species having a similar phytochemical composition. In this study, the effects of natural growth conditions (in situ) and ex situ cultivation and sampling locations on the phenolic compound contents and antioxidant activity of methanolic extracts of Salvia circinata from Oaxaca, Mexico, were investigated. Stem and young leaf samples were collected from plants growing in situ in two locations in Oaxaca and later from plants propagated clonally or vegetatively from propagules obtained in situ but grown ex situ. In both sets of samples, the contents of total polyphenols and flavonoids and the antioxidant activity were evaluated by spectrophotometry, and subsequently, the contents of phenols and specific flavonoids were identified and quantified by high-performance liquid chromatography with diode-array detection (HPLC–DAD). The growth conditions and locality of origin of the samples significantly influenced the contents of total polyphenols and flavonoids and antioxidant activity, with the in situ conditions in the locality of Reforma, Oaxaca, favoring higher levels. Two phenolic acids (chlorogenic and rosmarinic) and two flavonoids (isoquercitrin and rutin) were identified, and the concentrations of these compounds were influenced by the growth conditions and localities of origin of the samples.

Full text

Citation: Pérez-Ochoa, M.L.;
Vera-Guzmán, A.M.;
Mondragón-Chaparro, D.M.;
Sandoval-Torres, S.;
Carrillo-Rodríguez, J.C.;
Hernández-Delgado, S.;
Chávez-Servia, J.L. Plant and Growth
Condition Interactions on the
Phenolic Compound Contents and
Antioxidant Activity in Salvia
circinata Cav., a Medicinal Sage.
Diversity 2023,15, 656. https://
doi.org/10.3390/d15050656
Academic Editor: Michael Wink
Received: 5 April 2023
Revised: 8 May 2023
Accepted: 10 May 2023
Published: 12 May 2023
Copyright: © 2023 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/).
diversity
Article
Plant and Growth Condition Interactions on the Phenolic
Compound Contents and Antioxidant Activity in
Salvia circinata Cav., a Medicinal Sage
Mónica L. Pérez-Ochoa 1, Araceli M. Vera-Guzmán1,* , Demetria M. Mondragón-Chaparro 1,
Sadoth Sandoval-Torres 1, JoséC. Carrillo-Rodríguez 2, Sanjuana Hernández-Delgado 3
and JoséL. Chávez-Servia 1, *
1CIIDIR-Oaxaca, Instituto Politécnico Nacional, Santa Cruz Xoxocotlán, Oaxaca 71230, Mexico;
mperezo1800@alumno.ipn.mx (M.L.P.-O.); dmondragon@ipn.mx (D.M.M.-C.); ssandovalt@ipn.mx (S.S.-T.)
2Instituto Tecnológico del Valle de Oaxaca, Santa Cruz Xoxocotlán, Oaxaca 71230, Mexico;
jcarrillo_rodriguez@hotmail.com
3Centro de Biotecnología Genómica, Instituto Politécnico Nacional, Reynosa 88710, Mexico;
shernandezd@ipn.mx
*Correspondence: avera@ipn.mx (A.M.V.-G.); jchavezs@ipn.mx (J.L.C.-S.)
Abstract:
The genus Salvia has a worldwide distribution, and its contribution to traditional medicine
and as an aromatic plant has been recognized since ancient times, with few documented species
having a similar phytochemical composition. In this study, the effects of natural growth conditions
(in situ) and ex situ cultivation and sampling locations on the phenolic compound contents and
antioxidant activity of methanolic extracts of Salvia circinata from Oaxaca, Mexico, were investigated.
Stem and young leaf samples were collected from plants growing in situ in two locations in Oaxaca
and later from plants propagated clonally or vegetatively from propagules obtained in situ but
grown ex situ. In both sets of samples, the contents of total polyphenols and flavonoids and the
antioxidant activity were evaluated by spectrophotometry, and subsequently, the contents of phenols
and specific flavonoids were identified and quantified by high-performance liquid chromatography
with diode-array detection (HPLC–DAD). The growth conditions and locality of origin of the samples
significantly influenced the contents of total polyphenols and flavonoids and antioxidant activity,
with the in situ conditions in the locality of Reforma, Oaxaca, favoring higher levels. Two phenolic
acids (chlorogenic and rosmarinic) and two flavonoids (isoquercitrin and rutin) were identified, and
the concentrations of these compounds were influenced by the growth conditions and localities of
origin of the samples.
Keywords: ecological microniches; environmental effects; medicinal plants; bioactive compounds
1. Introduction
The genus Salvia (Lamiaceae) has a worldwide distribution and is widely used in
traditional medicine. Of particular interest is the subgenus Calosphace, which contains
587 species with a large number endemic to North and South America (500 species). The
countries with the highest diversity of Salvia are Mexico with 295 recognized species, Peru
with 77, Colombia with 60, and Brazil with 58 [
1
–
4
]. Many studies have documented
the ethnobotany, ethnomedicinal uses and chemical composition of the Salvia species
distributed in the Mediterranean region, Europe, Central Asia and South Africa [1,4,5].
Evaluations of the chemical composition and biological activity of Salvia species indi-
cate that they have pharmacological potential in treating gastrointestinal and respiratory
disorders and preventing neurodegenerative disorders. In addition, their antimicrobial,
antioxidant, antiinflammatory, cytotoxic and antispasmodic activities provide phytochem-
ical evidence supporting their use in traditional medicine [
6
–
10
]. Decoctions and infu-
sions of Salvia leaves and stems are used in the treatment of abdominal pain, vomiting,
Diversity 2023,15, 656. https://doi.org/10.3390/d15050656 https://www.mdpi.com/journal/diversity

Diversity 2023,15, 656 2 of 14
cardiac issues, cold, anxiety and gastrointestinal disorders and have preventive medic-
inal potential against diarrhea, diabetes, cancer, ulcers and helminthiasis [
10
–
12
]. The
effectiveness of species Salvia circinata is partly due to its composition. For example,
Moreno-Pérez et al. [13,14]
report that the antinociceptive and antiinflammatory medicinal
properties are conferred by the presence of the neo-clerodane terpene amarisolide A and the
flavonoid pedalitin. Flores-Bocanegra et al. [
11
] indicate that the antihyperglycemic action
in rat models is due to the inhibitory activity of the enzyme alpha-glucosidase. Additionally,
Salinas-Arellano et al. [
15
] note that the presence of flavonoids and terpenoids, such as the
flavonoids rutin, isoquercitrin and pedalitin, which have anxiolytic and/or antinociceptive
properties, inhibits the activity of protein tyrosine phosphatase 1B (PTP-1B).
Nurzy´nska-Wierdak [
16
] highlights that under natural growth conditions, the biosyn-
thesis of phenolic compounds in plants and their antioxidant and/or biological properties
are affected by ecological–environmental conditions, genotype (inherited ability to syn-
thesize metabolites) and ontogenetic variation, which are determinants of the synthesis
of a wide range of secondary metabolites. Changes in chemical profiles during ontogeny,
e.g., between different ages, organ types and plant phenological or developmental stages,
determine the chemical composition of a plant sample and help to determine the suitable
harvesting or sampling period. In wild medicinal plants, Bautista et al. [
17
] and
Li et al. [18]
agree that internal genetic factors related to development (e.g., regulatory genes and en-
zymes) and external environmental factors such as light, temperature, salinity and water
availability influence the synthesis, concentration and accumulation of antioxidant phenolic
compounds, and the effect of developmental and environmental factors changes depending
on the species.
Regarding the environmental effect on the phytochemical composition of Salvia species,
Bettaieb et al. [
19
] evaluated the effect of medium to severe water deficit on Salvia officinalis
and concluded that in general, drought increases the abundance of phenolic compounds,
but the concentration of specific compounds differs, with both significant increases and
decreases; for example, the concentrations of rosmarinic and chlorogenic acids increase,
while those of the flavonoids quercetin and quercetin 3-D-galactoside decrease. A similar
response was observed by Tavakoli et al. [
20
] when evaluating the effect of ecological factors
and soil characteristics on the concentration of phenols and flavonoids in S. multicaulis. The
authors concluded that the response differs by compound: with low temperatures, the con-
centration of total phenolic compounds increases, but cold stress reduces the concentration
of phenolic acids. Similar responses were observed for flavonoids when plants were under
prolonged stress caused by low rainfall, low relative humidity, low soil organic matter
content and low soil pH. That is, the set of growth conditions and/or plant genotypes of
Salvia species influence the contents of secondary metabolites. The objective of this study
was to evaluate the effects of natural (in situ) and cultivated (ex situ) growing conditions
on the contents of phenolic compounds and antioxidant activity in S. circinata, a medicinal
plant used in the Mixtec communities of Oaxaca, Mexico.
2. Materials and Methods
2.1. Plant Material
S. circinata Cav. (Syn. Salvia amarissima Ortega) is a perennial herbaceous plant that
reaches 0.3 to 1.5 m in height, with hispid stems and blue/purple inflorescences. The
species is endemic to Mexico, from San Luis Potosi to Oaxaca, at elevations ranging from
1650 to 2800 m. S. circinata grows preferentially in disturbed areas of temperate forests,
shrubs and grasslands [
21
,
22
]. This species is used in traditional Mexican medicine in
the treatment of gastrointestinal disorders (stomach pain, diarrhea, helminthiasis, ulcers)
and diabetes [
12
]. In San Martin Huamelulpam, Oaxaca (sampling region), S. circinata is
known as ‘yucucahua morada’. This species grows in places near forests and in disturbed
sites, such as on the margins of farm plots or roads and in patches near streams and water
sources during the rainy season (June–November). It is tolerated in backyards or orchards
as an ornamental plant. Additionally, it is applied as a medicine in the form of infusions of

Diversity 2023,15, 656 3 of 14
leaves and stems that are fresh, dried or chewed to cure or prevent stomach pain (‘latido’)
and diarrhea.
2.2. Experimental Design and Sampling
S. circinata was sampled based on a bifactorial design, where Factor A was the growth
condition (growth environment, E): plants growing in natural conditions (in situ) in the
municipality of San Martin Huamelulpam (Huamelulpam), Oaxaca, and plants grown in
ex situ conditions in Santa Cruz Xoxocotlan, Oaxaca, using propagules of the same plants
identified in situ that were assumed to be clones of the original plants. As part of the
design, Factor B was the site or localitions of ecogeographic origin of the populations in
their natural conditions: Reforma and La Union localities within the municipal territory of
San Martin Huamelulpam (population, Po). Therefore, combining the two levels of Factor
A with the two levels of Factor B, we designed an experiment with four experimental
combinations (A
×
B or E
×
Po). Table 1describes the geographic and environmental
conditions of the collection sites of S. circinata plants recollected in situ and cultivated ex situ.
Therefore, the in situ and ex situ sampling design was proposed to compare the populations
of plants growing under natural conditions versus the same plants propagated clonally or
vegetatively and grown under cultivation conditions as part of our statistical hypotheses.
Table 1. Geographic and environmental conditions of S. circinata sampled from Oaxaca, Mexico.
Geographic and Environmental
Growing Conditions of S. circinata
Natural Conditions (In Situ) Ex Situ Cultivation
San Martin Huamelulpam Santa Cruz
Xoxocotlan
Reforma La Union
Locality descriptors:
Altitude (m) 2151 2238 1530
Latitude (N) 17◦24024.00 0 17◦23030.80 0 17◦02064.60 0
Longitude (W) 97◦37011.50 0 97◦37026.30 0 96◦71095.30 0
Annual average temperature (◦C) a14–18 18–20
Annual average rainfall (mm) 700–1000 600–700
Solar radiation (W/m2, maximum) 1155–1308 1297–1349
Climate (predominant) Temperate subhumid with precipitation from June to
November Semidry to semiwarm
Soil chemical analysis b:
Organic matter (%) 4.67 7.15 9.41
pH (in H2O) 7.76 7.86 7.48
P-Olsen (mg kg−1)9.52 12.30 271.40
B-Olsen (mg kg−1)0.52 0.83 2.00
K (cmol kg−1)0.51 0.86 3.86
Ca (cmol kg−1)38.00 44.20 27.10
Mg (cmol kg−1)1.14 1.52 7.99
Na (cmol kg−1)0.16 0.35 1.83
Fe (mg kg−1)8.48 8.54 26.59
Zn (mg kg−1)2.02 2.40 56.22
Mn (mg kg−1)15.65 18.25 18.09
Cu (mg kg−1)0.59 0.70 1.75
Inorganic N (mg kg−1)6.78 16.80 283.15
Electric conductivity 0.27 0.40 2.28
Cation exchange capacity (cmol kg−1)41.00 48.00 41.50
aINEGI [23]; bSoil analysis under current Mexican norm [24].
For the experiment, the first sampling stage (in situ conditions) consisted of collecting
three samples of leaves and young stems from 10 to 15 individuals of S. circinata in a state
of flowering or incipient flowering (approximately 1 kg of fresh tissue per sample) at three
different sides of the population or from a ‘patch’ of plants in the locality of Reforma and
another similar sample at La Union, San Martin Huamelulpam, Oaxaca, between September
and October 2020 (after the rainy season). The second sampling stage corresponded to ex

Diversity 2023,15, 656 4 of 14
situ cultivation of plants grown from sections or propagules of S. circinata plants collected
from the same in situ sampling sites and cultivated inside a metal structure covered with
shade cloth from September 2020 to May 2021. The plants were grown in 20 L pots in
a substrate containing sand, soil and organic matter at a 1:1:1 ratio. Soil moisture was
maintained at field capacity, and the daily temperature and relative humidity were recorded.
Three pots were grown with plants from the Reforma locality and three pots from the La
Union locality. Three plant tissue samples from each pot were collected in May 2021 based
on the same criteria used for in situ sampling. In addition, soil and substrate samples were
taken from each field sampling site (in situ) and ex situ cultivation pot for chemical analysis
(Table 1).
2.3. Determination of Phenolic Compounds and Antioxidant Activity by Spectrophotometry
2.3.1. Sample Preparation
The in situ and ex situ plant material was washed, cut into small pieces, dried at
40
◦
C in a dehydrator (L’Equipe Model 528) and pulverized using a mill (Krups
®
model
GX4100, Mexico). Subsequently, samples were stored at
−
20
◦
C in an airtight container
until evaluation. For the spectrophotometric determinations of phenolic compounds and
antioxidant activity, a methanolic extract was obtained from 0.1 g of dry milled sample and
25 mL of 60% (v/v) methanol, which was homogenized for 60 s in intervals of 30 s (Ultra
Turrax T 25 Digital, IKA, Staufen, Germany) and centrifuged at 11,000 rpm (Eppendorf AG,
Model 5811F, Hamburg, Germany) at 4
◦
C for 15 min, and the supernatant was used for
the respective analyses. All determinations were carried out in triplicate.
2.3.2. Total Polyphenols
The analysis of total polyphenols was performed according to the Folin
–
Ciocalteu
method described by Singleton and Rossi [
25
], where the absorption was measured at
750 nm
using a spectrophotometer (VELAB, Model VE-5600UV PC, Pharr, TX, USA), and
the concentration was calculated with reference to a calibration curve of a gallic acid
standard (0.021 to 0.165 mg mL
−1
, r
2
= 0.999). The results were expressed as milligrams of
gallic acid equivalents per gram of dry weight (mg GAE g−1dw).
2.3.3. Flavonoids
The flavonoid contents were determined using two colorimetric methods. The first
method followed the methodology proposed by Lin and Tang [
26
], which uses the reaction
of aluminum chloride (AlCl
3
) in the presence of flavonoids to estimate the contents of
flavonols and flavones such as luteolin [
27
] as a function of absorbances at 415 nm in a
spectrophotometer. The concentration was estimated with reference to a standard quercetin
calibration curve (0.01 to 0.17 mg mL
−1
, r
2
= 0.999) and was expressed as milligrams of
quercetin equivalents per gram of dry weight (mg QE g
−1
dw). The second estimation
method involved the use of sodium nitrite (NaNO
2
) in an alkaline medium [
28
] to evaluate
the total contents of rutin, luteolin and catechins [
27
]. The absorbance at 510 nm was
recorded, and the estimation was quantified based on a standard catechin calibration curve
(0.01 to 0.5 mg mL
−1
, r
2
= 0.999). The results were expressed as milligrams of catechin
equivalents per gram of dry weight (mg CE g−1dw).
2.3.4. Antioxidant Activity Evaluated by DPPH and FRAP
The DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich, St. Louis, MO, USA)
method described by Brand-Williams et al. [
29
] was used. The absorbance was mea-
sured in a spectrophotometer at 517 nm, and the content was quantified with reference
to a Trolox calibration curve (0.13 to 1.33
µ
mol mL
−1
, r
2
= 0.999). Then, the antioxidant
activity was evaluated by the FRAP (ferric reducing antioxidant power) method based
on the description of Benzie and Strain [
30
]. In this case, the absorbance was recorded at
593 nm, and the concentration was calculated with reference to a Trolox calibration curve

Diversity 2023,15, 656 5 of 14
(0.05 to 1.0
µ
mol mL
−1
, r
2
= 0.999). The results of both determinations were expressed as
micromoles of Trolox equivalents per gram of dry weight (µmol TE g−1dw).
2.4. Determination of Phenolic Acids and Flavonoids by HPLC–DAD
A methanolic extract was obtained from 0.2 g of dry milled sample and 10 mL of
60% (v/v) methanol, which was homogenized for 60 s in intervals of 30 s (Ultra Turrax
T 25 Digital, IKA, Staufen, Germany) and sonicated in an ultrasonic bath (Cole-Parmer,
Model 08895-43, Vernon Hills, IL, USA) for 1 h. Subsequently, the sample was centrifuged
at
11,000 rpm
(refrigerated Eppendorf 5810R centrifuge) at 4
◦
C for 15 min, and the su-
pernatant was used for HPLC–DAD analysis. Then, a 2 mL aliquot of methanolic extract
(60%) was filtered by a 0.2
µ
m PTFE syringe (Agilent Technologies
®
, Part. No. 5190-5086,
Waldbronn, Germany) and placed in amber vials.
The analysis of phenolic acids and flavonoids was performed using HPLC–DAD
following the method described by Paj ˛ak et al. [
31
] with some modifications described
in a previous study [
32
]. The instrument was an HPLC (Agilent model Infinity II 1260
LC system) equipped with a solvent degasser, quaternary pump, temperature-controlled
autosampler, column oven and DAD (Agilent Technologies, Santa Clara, CA, USA). A
reversed-phase column (Agilent
®
Hypersil 5 ODS, 250
×
4.6 mm, 5
µ
m) was used. The
sample analysis characteristics and conditions were described in Perez-Ochoa et al. [
32
],
where the monitoring wavelengths for flavonoids and phenolic acids were 260 nm (rutin
and isoquercitrin) and 320 nm (chlorogenic and rosmarinic acids), respectively. The identi-
fication of each compound was made with reference to the retention times and UV spectra
of commercial standards (Phyproof
®
, Phytolab GmbH & Co. KG, Vestenbergsgreuth, Ger-
many, and Sigma-Aldrich
®
, St. Louis, MO, USA) and quantification by calibration curves
of reference standards: chlorogenic acid (1.27 to 203
µ
g mL
−1
, r
2
= 0.998), rosmarinic acid
(0.9 to 533
µ
g mL
−1
, r
2
= 0.999), rutin (0.9 to 256
µ
g mL
−1
, r
2
= 0.999) and isoquercitrin (0.5
to 72
µ
g mL
−1
, r
2
= 0.999). The amount of each compound was expressed as micrograms
per gram of dry weight (µg g−1dw).
2.5. Statistical Analysis
Based on the information obtained from the phytochemical analysis of the samples,
a database was established, and analysis of variance was performed using a completely
random linear model. To evaluate the specific differences between localities of sample
origin (Po, populations), between plant growth environments (E, in situ and ex situ) and
among locality–environment interactions (Po
×
E), Tukey’s test (p< 0.05) was used for
means comparisons. In addition, Pearson correlation analysis was performed between
total polyphenols and flavonoids versus antioxidant activity, and principal component
analysis was conducted using the average values per sample. All statistical analyses were
performed with the SAS statistical package (SAS Institute Inc., Cary, NC, USA) [33].
3. Results
3.1. Total Phenolic Compounds and Antioxidant Activity
In the analysis of variance, significant differences in the contents of total polyphe-
nols and flavonoids and antioxidant activity were observed (p< 0.01) between growth
conditions (E) and between localities of origin of the samples or populations (Po). For the
interaction between growth conditions and populations (E
×
Po), significant differences
were recorded only for antioxidant activity evaluated by the FRAP method, indicating
that for the concentration of polyphenols and flavonoids and antioxidant activity as deter-
mined by the DPPH method, the effect of growth conditions is independent of the effect of
populations. The results of the analysis of variance showed that the variance due to the
locality of origin of the samples or population (Po) was significantly greater than the effect
of growth condition (E) and E
×
Po interactions on the content of phenolic compounds and
antioxidant activity (Table 2).

Diversity 2023,15, 656 6 of 14
Table 2.
Significance of the mean squares of the analysis of variance of phenolic compounds and
antioxidant activity in S. circinata from San Martin Huamelulpam, Oaxaca, Mexico.
Sources of Variation Total Polyphenols Flavonoids Antioxidant Activity
Quercetin Catechin DPPH FRAP
Growth environment (E)
2113.8 ** 420.4 ** 2797.9 ** 193,044.6 ** 349,462.7 **
Locality of origin (Po) 8230.3 ** 421.7 ** 4991.2 ** 439,294.9 ** 711,823.6 **
E×Po 31.8 ns 0.02 ns 78.9 ns 4928.2 ns 38,766.0 **
Sampling (S) 154.7 ns 9.0 ns 18.9 ns 831.1 ns 18,120.1 *
Lab. replicate/S 137.0 ns 3.0 ns 16.6 ns 42.2 ns 1012.3 ns
Error 9.6 0.8 2.7 163.7 828.2
Coeff. of variation (%) 4.3 5.9 3.2 3.0 4.0
** Significant at p< 0.01; * Significant at p< 0.05;
ns
= not significant (p> 0.05).
1
Indicates nesting of laboratory
replicates within sampling.
In the comparison of the means of the effect of the growth conditions (in situ vs. ex
situ), the response pattern showed that in all the parameters evaluated, the plants grown
in natural conditions had significantly higher concentrations of phenolic compounds and
antioxidant activity than those grown ex situ. Analysis of the effect of the localities of origin
of the samples (Po) showed that the leaves and stems collected in the locality of Reforma
had significantly higher concentrations of polyphenols, flavonoids and antioxidant activity
than those collected in La Union, Huamelulpam (Table 3).
Table 3.
Average content of phenolic compounds and antioxidant activity of S. circinata plants
sampled in situ (natural conditions) and cultivated ex situ in Oaxaca, Mexico.
Study Factors Levels Total Polyphenols
(mg GAE g−1dw)
Flavonoids
(mg g−1dw)
Antioxidant Activity
(µmol TE g−1dw)
QE 1CE 1DPPH FRAP
Growth
environment (E)
In situ 78.0 ±12.6 a 217.8 ±3.3 a 58.9 ±8.5 a 486.5 ±62.5 a 806.4 ±121.2 a
Ex situ 69.1 ±11.8 b 13.9 ±3.8 b 48.7 ±8.4 b 402.0 ±88.3 b 692.6 ±99.3 b
Localities
of origin (Po) Reforma 79.8 ±10.4 a 216.8 ±4.4 a 57.6 ±8.6 a 490.3 ±69.2 a 792.7 ±101.4 a
La Union 62.9 ±7.9 b 12.9 ±2.3 b 44.9 ±4.8 b 356.0 ±49.5 b 649.3 ±79.4 b
Environment–locality (E ×Po) interactions:
Reforma In situ 87.2 ±8.3 a 219.8 ±3.4 a 66.5 ±2.7 a 543.6 ±23.4 a 906.5 ±52.8 a
Ex situ 77.3 ±9.9 a 15.8 ±4.3 a 54.6 ±7.9 a 472.5 ±70.4 a 754.8 ±83.8 b
La Union In situ 68.7 ±8.5 a 15.8 ±1.5 a 51.2 ±4.1 a 429.5 ±24.8 a 706.2 ±78.9 b
Ex situ 60.9 ±6.7 a 11.9 ±1.5 a 42.7 ±2.7 a 331.5 ±25.3 a 630.3 ±70.7 c
1
QE and CE, quercetin and catechin equivalents, respectively;
2
In columns, means between environments,
localities or environment–locality interactions followed by the same letter are not different significantly (Tukey’s
test, p< 0.05).
The interaction between localities of origin and growth conditions did not contribute
significantly to differences in total polyphenols and flavonoids or antioxidant activity
evaluated by DPPH; however, there was a significant difference in antioxidant activity
as determined by FRAP (Table 3). This indicates independence of the study factors (Po
and E) but with similar effect in magnitude and significantly greater than the effect of
their interaction. These results suggest similar compound contents or antioxidant activity
and reflect the fact that the magnitude of the effect of the growth conditions (ontogenetic
and ecological factors) is similar and independent of the effect of the locality of origin of
the samples.
Pearson’s correlation analysis revealed a significant and positive relationship between
total polyphenols and catechin and quercetin equivalent flavonoids with antioxidant ac-
tivity evaluated by the DPPH and FRAP methods in S. circinata plants collected in situ
(0.68 < r < 0.97;
p< 0.001) and cultivated ex situ (0.74 < r < 0.95; p< 0.001). In both cases,
the phenolic compounds evaluated contributed substantially to the antioxidant activity (as
determined by the DPPH and FRAP methods) in methanolic extracts of S. circinata.

Diversity 2023,15, 656 7 of 14
3.2. Profile of Phenolic Acids and Flavonoids by HPLC–DAD
In the HPLC–DAD analysis of phenolic compounds in methanolic extracts of leaves
and young stems of S. circinata, two phenolic acids, chlorogenic and rosmarinic acids, and
two glycosylated flavonoids derived from quercetin, rutin (quercetin-3-rutinoside) and
isoquercitrin (quercetin-3-β-D-glucoside), were identified and quantified (Figure 1).
Diversity 2023, 15, x FOR PEER REVIEW 7 of 14
Table 3. Average content of phenolic compounds and antioxidant activity of S. circinata plants sam-
pled in situ (natural conditions) and cultivated ex situ in Oaxaca, Mexico.
Study Factors
Levels
Total Polyphenols
(mg GAE g−1 dw)
Flavonoids
(mg g−1 dw)
Antioxidant Activity
(µmol TE g−1 dw)
QE 1
CE 1
DPPH
FRAP
Growth
environment (E)
In situ
78.0 ± 12.6 a 2
17.8 ± 3.3 a
58.9 ± 8.5 a
486.5 ± 62.5 a
806.4 ± 121.2 a
Ex situ
69.1 ± 11.8 b
13.9 ± 3.8 b
48.7 ± 8.4 b
402.0 ± 88.3 b
692.6 ± 99.3 b
Localities
of origin (Po)
Reforma
79.8 ± 10.4 a 2
16.8 ± 4.4 a
57.6 ± 8.6 a
490.3 ± 69.2 a
792.7 ± 101.4 a
La Union
62.9 ± 7.9 b
12.9 ± 2.3 b
44.9 ± 4.8 b
356.0 ± 49.5 b
649.3 ± 79.4 b
Environment–locality (E × Po) interactions:
Reforma
In situ
87.2 ± 8.3 a 2
19.8 ± 3.4 a
66.5 ± 2.7 a
543.6 ± 23.4 a
906.5 ± 52.8 a
Ex situ
77.3 ± 9.9 a
15.8 ± 4.3 a
54.6 ± 7.9 a
472.5 ± 70.4 a
754.8 ± 83.8 b
La Union
In situ
68.7 ± 8.5 a
15.8 ± 1.5 a
51.2 ± 4.1 a
429.5 ± 24.8 a
706.2 ± 78.9 b
Ex situ
60.9 ± 6.7 a
11.9 ± 1.5 a
42.7 ± 2.7 a
331.5 ± 25.3 a
630.3 ± 70.7 c
1 QE and CE, quercetin and catechin equivalents, respectively; 2 In columns, means between envi-
ronments, localities or environment–locality interactions followed by the same leer are not dier-
ent signicantly (Tukey’s test, p < 0.05).
Pearson’s correlation analysis revealed a signicant and positive relationship be-
tween total polyphenols and catechin and quercetin equivalent avonoids with antioxi-
dant activity evaluated by the DPPH and FRAP methods in S. circinata plants collected in
situ (0.68 < r < 0.97; p < 0.001) and cultivated ex situ (0.74 < r < 0.95; p < 0.001). In both cases,
the phenolic compounds evaluated contributed substantially to the antioxidant activity
(as determined by the DPPH and FRAP methods) in methanolic extracts of S. circinata.
3.2. Prole of Phenolic Acids and Flavonoids by HPLC–DAD
In the HPLC–DAD analysis of phenolic compounds in methanolic extracts of leaves
and young stems of S. circinata, two phenolic acids, chlorogenic and rosmarinic acids, and
two glycosylated avonoids derived from quercetin, rutin (quercetin-3-rutinoside) and
isoquercitrin (quercetin-3-β-D-glucoside), were identied and quantied (Figure 1).
Figure 1. Chromatographic prole based on HPLC–DAD analysis of phenolic compounds in meth-
anolic extracts from leaves and young stems of S. circinata (a) and based on reference standards (b)
(1) chlorogenic acid, (2) rutin, (3) isoquercitrin and (4) rosmarinic acid.
Figure 1.
Chromatographic profile based on HPLC–DAD analysis of phenolic compounds in methano-
lic extracts from leaves and young stems of S. circinata (
a
) and based on reference standards (
b
)
(1) chlorogenic acid, (2) rutin, (3) isoquercitrin and (4) rosmarinic acid.
The analysis of variance showed significant differences (p< 0.05, 0.01) in isoquercitrin
and rutin between growth environments (E), between localities of origin of the samples or
populations (Po) and among the environment–locality interactions (E
×
Po). However, for
chlorogenic acid, significant differences were recorded between populations and due to
the E
×
Po interaction, and in rosmarinic acid, differences were observed between growth
conditions (E) and due to the E
×
Po interaction. The results show that phenolic acids
and flavonoids have differential response patterns depending on the study factors. For
example, for chlorogenic acid and isoquercitrin, the variance due to the locality of origin of
the samples was significantly greater than that due to the growth conditions or the E
×
Po
interaction; for rosmarinic acid, the variance due to the effect of the E
×
Po interaction and
growth conditions similarly influenced the concentration; and for rutin, the factor with the
greatest influence on variance was the growth conditions (Table 4).
Table 4.
Significance of mean squares of the analysis of variance of phenolic acids and flavonoids
identified in S. circinata by HPLC–DAD.
Sources of Variation Chlorogenic Acid Rosmarinic Acid Isoquercitrin Rutin
Growth environment (E) 79.0 ns 900.9 * 1046.9 ** 178,322,423 **
Localities of origin (Po) 7711.8 ** 41.8 ns 3389.9 ** 80,878,246 **
E×Po 2993.4 * 953.5 * 270.2 ** 42,704,363 **
Sampling (S) 1995.4 ** 103.5 ns 46.1 ns 21,168,781 **
Rep./S 1<0.01 ns 117.8 ns 0.04 ns 161,649 ns
Error 527.4 168.4 12.6 367,122.3
Coeff. variation (%) 23.2 13.6 16.8 12.9
1
Laboratory replicates nested within in situ and ex situ sampling;
ns
not significant (p> 0.05); * significant at
p< 0.05; ** significant at p< 0.01.

Diversity 2023,15, 656 8 of 14
The S. circinata plants collected under natural growing conditions (in situ) presented
significantly higher contents of rosmarinic acid, isoquercitrin and rutin than the plants
cultivated ex situ, but the contents of chlorogenic acid did not differ significantly. The
comparison of means between localities of origin of the samples (Po) showed two con-
trasting patterns. First, for the contents of chlorogenic acid and rutin, the samples from
Reforma presented higher values than the samples from La Union. For the isoquercitrin
content, the opposite pattern was observed, with significantly higher contents in samples
from La Union than from Reforma (Table 5). That is, the growth conditions and localities of
origin of the samples differentially influenced the composition of phenols and flavonoids
in S. circinata.
Table 5.
Phenolic acid and flavonoid contents identified by HPLC–DAD in S. circinata sampled in
situ (natural conditions) and cultivated ex situ in Oaxaca, Mexico.
Compounds
Evaluated
(µg g−1dw)
Growth Environments (E) Localities of Origin
(Po)
Locality–Environment Interactions (Po ×E)
Reforma La Union
In Situ Ex Situ Reforma La Union In Situ Ex Situ In Situ Ex Situ
Chlorogenic acid 100.8 a 198.0 a 112.5 a 185.2 b 105.6 a 1117.1 a 95.6 b 78.9 b
Rosmarinic acid 10,451.1 a 8690.4 b 9546.5 a 9242.8 a 11,677.5 a 8125.8 b 9224.7 b 9254.9 b
Isoquercitrin 797.5 a 353.4 b 198.7 b 863.5 a 272.2 c 149.7 c 1322.8 a 557.2 b
Rutin 7668.4 a 2775.8 b 5697.4 a 3633.8 b 9585.5 a 3105.4 c 5577.1 b 2446.2 c
1
Within rows, means between environments, localities or locality–environment interactions followed by the same
letter are not significantly different (Tukey’s test, p< 0.05).
The concentrations of chlorogenic acid, rosmarinic acid, isoquercitrin and rutin in
methanolic extracts of S. circinata were influenced by the growth conditions or environments
and populations or sample origins (E
×
Po), but the response pattern differed by compound.
For example, for chlorogenic acid, the in situ and ex situ samples from the Reforma locality
showed a higher concentration than the samples from La Union, indicating that the effect
of locality was significant. For isoquercitrin content, the opposite effect of locality was
observed. Additionally, the concentration patterns of rosmarinic acid and rutin differed,
where the Reforma samples collected in situ (natural conditions) presented higher values
than those cultivated ex situ and those originating from the La Union locality (in situ and
ex situ) (Table 5).
In the principal component analysis (PCA), the S. circinata samples showed two
dispersion patterns based on their composition of phenolic compounds and antioxidant
activity (Figure 2). The samples of leaves and young stems of S. circinata collected in situ
presented a pattern of high dispersion associated with the rosmarinic acid and rutin contents
and antioxidant activity, and in contrast, the ex situ samples were less dispersed. In Figure 2,
the natural conditions or in situ environment of origin locations influenced the dispersion
and/or differentiation between Reforma and La Union. Therefore, it seems necessary to
consider the effect of sites and locations on the multivariate differences between samples.

Diversity 2023,15, 656 9 of 14
Diversity 2023, 15, x FOR PEER REVIEW 9 of 14
Reforma locality showed a higher concentration than the samples from La Union, indicat-
ing that the eect of locality was signicant. For isoquercitrin content, the opposite eect
of locality was observed. Additionally, the concentration paerns of rosmarinic acid and
rutin diered, where the Reforma samples collected in situ (natural conditions) presented
higher values than those cultivated ex situ and those originating from the La Union local-
ity (in situ and ex situ) (Table 5).
In the principal component analysis (PCA), the S. circinata samples showed two dis-
persion paerns based on their composition of phenolic compounds and antioxidant ac-
tivity (Figure 2). The samples of leaves and young stems of S. circinata collected in situ
presented a paern of high dispersion associated with the rosmarinic acid and rutin con-
tents and antioxidant activity, and in contrast, the ex situ samples were less dispersed. In
Figure 2, the natural conditions or in situ environment of origin locations inuenced the
dispersion and/or dierentiation between Reforma and La Union. Therefore, it seems nec-
essary to consider the eect of sites and locations on the multivariate dierences between
samples.
Figure 2. Dispersion of S. circinata samples by locality of origin and growth condition, based on the
rst two principal components of the analysis of phenolic compounds.
4. Discussion
The spectrophotometric analysis of methanolic extracts of S. circinata consistently
showed that the samples of plants growing in their natural conditions (in situ) presented
higher contents of polyphenols and avonoids and higher antioxidant activity than plants
grown ex situ (Table 2). This dierential response to in situ and ex situ conditions reects
part of the factors that induce stress in S. circinata plants and suggests that the biosynthesis
of metabolites is the product of a complex of ecophysiological and metabolic responses to
stress conditions. For example, in ex situ conditions, the plants grew without moisture
restrictions, and substrates had higher contents of organic maer and micro- and macro-
nutrients than the plants that grew in situ, which were subject to the rainy season, possible
stress due to water decit or drought, lower contents of soil nutrients and lower temper-
atures (Table 1). The elements and factors that cause abiotic stress were noted by Arbona
Figure 2.
Dispersion of S. circinata samples by locality of origin and growth condition, based on the
first two principal components of the analysis of phenolic compounds.
4. Discussion
The spectrophotometric analysis of methanolic extracts of S. circinata consistently
showed that the samples of plants growing in their natural conditions (in situ) presented
higher contents of polyphenols and flavonoids and higher antioxidant activity than plants
grown ex situ (Table 2). This differential response to in situ and ex situ conditions reflects
part of the factors that induce stress in S. circinata plants and suggests that the biosynthesis
of metabolites is the product of a complex of ecophysiological and metabolic responses
to stress conditions. For example, in ex situ conditions, the plants grew without mois-
ture restrictions, and substrates had higher contents of organic matter and micro- and
macronutrients than the plants that grew in situ, which were subject to the rainy season,
possible stress due to water deficit or drought, lower contents of soil nutrients and lower
temperatures (Table 1). The elements and factors that cause abiotic stress were noted by
Arbona et al. [
34
] and Verma and Shukla [
35
] as responsible for inducing fluctuations in the
concentration of secondary metabolites in plants.
Arbona et al. [
34
] and Yang et al. [
36
] emphasized that drought or water stress is
one of the main limiting factors for the growth, reproductive development and survival
of plants because it affects the photosynthetic capacity, nutrient absorption, osmotic reg-
ulation and other metabolic processes in plants that require water. In Salvia multicaulis,
Tavakoli et al. [20]
estimated a direct relationship between the low temperatures of the
sampled regions and increases in total polyphenols and a positive relationship between
flavonoid contents and sampling regions where there was limited rainfall and low relative
humidity, soil organic matter content and soil pH. Bettaieb et al. [
19
] experimentally showed
that severe drought induced a lower concentration of flavonoids and antioxidant activity
in S. officinalis plants than in controls for which there were no moisture restrictions. This
indicates that, under natural or experimental conditions (i.e., in cultivation), responses
differ by species, and changes in the synthesis of phenolic compounds and antioxidant
activity depend on the abiotic factor and severity or duration of stress.
Under natural conditions, S. circinata grows in forests and disturbed sites of San Martin
Huamelulpam (sampling region) and in patches near streams and water sources during the

Diversity 2023,15, 656 10 of 14
rainy season (June–November) and sprouts and flowers every year from December to May.
In April or May, there are low temperatures in this region (<18
◦
C), with frosts from Novem-
ber to January, and because there is little or no precipitation, there are no new shoots at this
time. The localities sampled within the target municipality for this work were Reforma
and La Union, and the distance between sampling sites was less than 3 km. Therefore,
there is no reason to believe that the sampled plants were from independent populations
but rather were subpopulations growing in different ecogeographic conditions or patches
because this region is highly disturbed. In this context of disturbances and anthropogenic
influences, the higher polyphenol and flavonoids concentrations and antioxidant activity in
the samples collected in Reforma than in the samples collected in La Union is due, to a large
extent, to differences in ecological, edaphic and microenvironmental or niche conditions
between sampling localities (Table 3).
The differences in polyphenols, flavonoids and antioxidant activity between S. circinata
collection sites are similar to the responses estimated by Farhat et al. [
37
] for the total phenol
content and antioxidant activity in S. officinalis,S. verbenaca,S. aegyptiaca and S. argentea
collected from sites with contrasting bioclimatic characteristics in Tunisia. Sarrou et al. [
38
]
recorded similar significant differences in phenol and total flavonoid contents between
two cultivated populations of S. fruticosa evaluated monthly from April to October (in a
warm environment with high relative humidity), emphasizing that the differences between
populations suggest genotypic differences and the influence of the harvest season. In this
work, pH, P, Mn, and inorganic N were lower in Reforma than in La Union (Table 1). In
addition to these differences, possible genetic differences generated by genetic drift due to
natural selection pressures and anthropic disturbances cannot be excluded. The suggestion
of genetic differences is based on the phenotypic model proposed by Lynch and Walsh [
39
],
where plant phenotype is a result of genotypic, environmental and genotype–environment
interaction effects (P = G + E + G
×
E), which is similar to the analysis model used in
this work.
The absence of significant interaction effects between localities of origin and growth
conditions (in situ and ex situ) on the content of total polyphenols, flavonoids (QE and
CE) and antioxidant activity as evaluated by DPPH suggests that both factors act or
influence each other independently. However, phenolic compounds showed a significant
and positive correlation with antioxidant activity in the samples collected in situ and those
from ex situ cultivation (0.68 < r > 0.97; p< 0.001). These results indicate that the samples
consist of complex matrices of compounds; for example, in Salvia fruticosa, Sarrou et al. [
38
]
identified 28 mono- and sesquiterpenes and 38 phenolic compounds, all with greater or
lesser influence on antioxidant activity. Therefore, the medicinal use of S. circinata in Mexico
and its positive effect against gastrointestinal disorders through decoctions or infusions is
based on the complex of ingested compounds.
In extracts of S. circinata, compounds such as pedalitin, apigenin-7-O-
β
-D-glucoside,
flavone 2-(3,4-dimethoxyphenyl)-5,6-dihydroxy-7-methoxy-4H-chromen-4-one, apigenin,
5,6-dihydroxy-7,3
0
,4
0
-trimethoxy-flavone, 5,6,4
0
-trihydroxy-7,3
0
-dimethoxyflavone,
6-hydroxyluteolin, rutin, isoquercitrin and rosmarinic acid have been
identified [10,11,13,15].
In this study, the contents of chlorogenic acid, rosmarinic acid, rutin and isoquercitrin were
identified and quantified in methanolic extracts of leaves and young stems of S. circinata,
and the effects of sample locality of origin (Po, populations), growth environment (E) and
locality–environment interaction (Po ×E) and variations in the response pattern between
compounds were identified (Table 4).
Chlorogenic acid is common in tissues of the genus Salvia, as previously reported in
S. officinalis by Bettaieb et al. [
19
], in S. fruticosa by Sarrou et al. [
38
] and in S. multicaulis
by Tavakoli et al. [
20
]. In this study, the concentration of chlorogenic acid in S. circinata
was significantly influenced by the locality of origin (Po) of the sample and the interaction
between growth environments and the locality of origin (E
×
Po). The plants collected in
Reforma had higher concentrations than their counterparts from La Union. Based on the
E×Po
interaction, the plants grown in situ or ex situ in Reforma had higher concentrations

Diversity 2023,15, 656 11 of 14
than those grown in La Union. These results suggest that in Reforma, there are populations
of S. circinata with chlorogenic acid biosynthetic characteristics that differ (e.g., chemotype)
from those in the La Union samples, and the greater effect of site or locality than of
growth conditions on this compound is evident among the plants grown in situ and ex
situ (Tables 4and 5). The results of Sarrou et al. [
38
] show that the sampling site and
genotype in cultivated S. fruticosa exert a strong effect on the chlorogenic acid content, but
Bettaieb et al. [
19
] noted that severe drought exerts an increasing effect on the chlorogenic
acid content in S. officinalis. This indicates that environmental or seasonal and genetic or
populational factors induce changes in chlorogenic acid biosynthesis, and the effects differ
by species.
Rosmarinic acid is characteristic of the family Lamiaceae and one of the main con-
stituents of the Salvia genus, and its potential antioxidant characteristics are associated with
anticancer, antibacterial, antiviral and antidiabetic properties [
5
,
40
,
41
]. In this study, the
rosmarinic acid content in S. circinata was influenced by the effect of growth conditions (E)
and the interaction between the growth environment and locality of origin of the samples
(E ×Po).
The results show that the natural conditions (in situ) have a significant effect on
the biosynthesis and accumulation of this compound since the concentration in situ ex-
ceeded the concentration in plants cultivated ex situ by 20%; this pattern is repeated for the
E
×
Po interaction, with the concentration of rosmarinic acid in the samples collected in situ
in Reforma exceeding that estimated in ex situ samples from Reforma and in all samples
originating from La Union by 26 to 43% (Table 5). Zengin et al. [
42
] noted that the ros-
marinic acid content differed between the Salvia species S. euphratica,S. blepharochlaena and
S. verticillata, and sampling site influences these differences, as shown by Farhat et al. [37]
in four species of sage. For Sarrou et al. [
38
], the sampling site was the main determin-
ing factor of the concentration of rosmarinic acid in S. fruticosa, which contrasts with the
results of Bettaieb et al. [
19
], who noted that drought or moderate water stress favors a
higher concentration of this compound since severe drought induces growth. Therefore,
the results suggest that growth conditions and moderate stress favor the biosynthesis and
accumulation of rosmarinic acid in the leaves and young stems of different species of sage.
Rutin and isoquercitrin are antioxidant glycosylated flavonoids present in some species
of Salvia, such as S. fruticosa [
38
], Salvia miltiorrhiza and S. amarissima from Oaxaca, Mex-
ico [
15
]. The previously identified patterns of chlorogenic and rosmarinic acid contents are
confirmed, in some way or in part. First, the concentrations of rutin and isoquercitrin were
significantly higher in samples collected in situ than ex situ, but the contents differed by
locality of origin. A higher concentration of rutin was estimated in samples from Reforma
than in samples from La Union, and for isoquercitrin, the opposite pattern was recorded
(La Union > Reforma). These results were confirmed by analyzing the interaction between
growth environments and localities of origin. The isoquercitrin concentration was higher
in samples collected in situ in La Union, while for rutin, the highest concentration was
recorded in in situ samples from Reforma (Table 5). Therefore, the results show that plants
growing in natural conditions (in situ) produce higher contents of isoquercitrin and rutin,
but this also depends on the locality or site of origin of the samples.
The variation in the pattern of isoquercitrin and rutin concentrations in S. circinata
shows that the ecological–environmental and geographic or site conditions, including
probable interpopulation genetic differences, generated some stress and a greater increase
in the biosynthesis and accumulation of these flavonoids in leaves and stems. For exam-
ple, severe water stress or drought leads to the excessive production of reactive oxygen
species, followed by oxidative stress and decreases in photosynthesis, and induces an
increase in flavonoid contents as part of the responses of plants to counteract metabolic
damage [19,34,43].
5. Conclusions
The spectrophotometric analysis of total polyphenols, flavonoids and antioxidant
activity in methanolic extracts of leaves and young stems of S. circinata collected under

Diversity 2023,15, 656 12 of 14
natural conditions (in situ) and cultivated ex situ showed that the ecological–environmental
and edaphic conditions of in situ growth and possible stress conditions significantly in-
fluenced the biosynthesis and accumulation of greater amounts of phenolic compounds
and led to higher antioxidant activity than that observed without water restriction and
with the provision of fertile substrate. In addition, the locality of origin of the samples
significantly influenced the concentrations analyzed, even when the distance between the
collection sites (Reforma and La Union) was less than 3 km. In the HPLC–DAD analysis,
two phenolic acids (chlorogenic acid and rosmarinic acid) and two flavonoids (rutin and
isoquercitrin) were identified with differential response patterns to the evaluated factors
(growth environment and locality of origin of the sampled population) and their interaction.
In this study, the concentration of chlorogenic acid was fundamentally influenced by the
origin of the sample (Reforma > La Union). For rosmarinic acid, rutin and isoquercitrin,
the in situ growth conditions were very influential in inducing a higher concentration than
that in the ex situ plants. Rutin and isoquercitrin had higher concentrations under in situ
conditions and showed opposite responses according to the locality of origin. The different
patterns in the synthesis and concentration of specific phenolic compounds suggest the
presence of different chemotypes or populations of S. circinata and help to partly explain
the medicinal properties of the species for the treatment of gastrointestinal disorders.
Author Contributions:
Conceptualization and methodology, M.L.P.-O., A.M.V.-G., D.M.M.-C., S.S.-T.,
J.C.C.-R. and J.L.C.-S.; investigation and writing, M.L.P.-O., A.M.V.-G., S.H.-D. and J.L.C.-S. All
authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by Instituto Politecnico Nacional-Mexico under projects no.
SIP-20231194 and SIP-20230580.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors thank all families from the municipalities of San Martin Huamelul-
pam, Oaxaca, Mexico, who shared their experiences with medicinal plants. In addition, we appreciate
the correction of the taxonomic classification of the plant species made by Rosalinda Medina-Lemos
of the Institute of Biology, Universidad Nacional Autonoma de Mexico.
Conflicts of Interest: The authors declare no conflict of interest.
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