plants
Article
Dynamics of Polyphenol Biosynthesis by Calli Cultures,
Suspension Cultures and Wild Specimens of the Medicinal
Plant Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae).
Analysis of Their Biological Activity
María Valeria Ricco 1,2 , Martín León Bari 1,2 , Alejandra Vanina Catalano 3,4 , Paula López 3,4 ,
Cecilia Beatriz Dobrecky 5,6 , Sergio Adrián Teves 7,8 , Ariana Posadaz 9 , Melina Laguia Becher 1,2 ,
Rafael Alejandro Ricco 5 , Marcelo Luis Wagner 5 and María Alejandra Álvarez 1,2, *
1
2
Citation: Ricco, M.V.; Bari, M.L.;
3
4
Catalano, A.V.; López, P.; Dobrecky,
C.B.; Teves, S.A.; Posadaz, A.; Laguia
5
Becher, M.; Ricco, R.A.; Wagner, M.L.;
et al. Dynamics of Polyphenol
Biosynthesis by Calli Cultures,
6
Suspension Cultures and Wild
Specimens of the Medicinal Plant
Ligaria cuneifolia (Ruiz & Pav.) Tiegh.
(Loranthaceae). Analysis of Their
7
8
9
Biological Activity. Plants 2021, 10,
1713. https://doi.org/10.3390/
*
Centro de Estudios Biomédicos, Básicos, Aplicados y Desarrollo (CEBBAD), Facultad de Ciencias de la Salud,
Universidad Maimónides, Hidalgo 775, Ciudad Autónoma de Buenos Aires 1405, Argentina;
ricco.mariavaleria@maimonides.edu (M.V.R.); bari.martin@maimonides.edu (M.L.B.);
melinalb@gmail.com (M.L.B.)
Consejo Nacional de Investigaciones Científicas y Técnicas, Godoy Cruz 2290,
Ciudad Autónoma de Buenos Aires 1425, Argentina
Cátedra de Farmacognosia, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956,
Ciudad Autónoma de Buenos Aires 1113, Argentina; alejandracatalano@gmail.com (A.V.C.);
plopez@ffyb.uba.ar (P.L.)
CONICET, Instituto de la Química y Metabolismo del Fármaco (IQUIMEFA), Junín 956,
Ciudad Autónoma de Buenos Aires 1113, Argentina
Cátedra de Farmacobotánica, Departamento de Farmacología, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Junín 956, Ciudad Autónoma de Buenos Aires 1113, Argentina;
cecilia.dobrecky@gmail.com (C.B.D.); raricco@gmail.om (R.A.R.); mlwagner@ffyb.uba.ar (M.L.W.)
Departamento de Tecnología Farmacéutica, Cátedra de Tecnología Farmacéutica I, Facultad de Farmacia
y Bioquímica, Universidad de Buenos Aires, Junín 956, Ciudad Autónoma de Buenos Aires 1113, Argentina
Cátedra de Microbiología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956,
Ciudad Autónoma de Buenos Aires 1113, Argentina; steves@proanalisis.com.ar
Proanálisis S.A, Av. San Martin 2355, Ciudad de Buenos Aires 1416, Argentina
Facultad de Turismo y Urbanismo, Universidad Nacional de San Luis, Av. del Libertador s/n,
Barranca Colorada, Villa de Merlo, San Luis 5881, Argentina; arianaposadaz@yahoo.com.ar
Correspondence: alvarez.mariaalejandra@maimonides.edu
plants10081713
Academic Editor: Anna Berim
Received: 12 July 2021
Accepted: 12 August 2021
Published: 20 August 2021
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Abstract: Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is a South American hemiparasitic species
with antioxidant, antitumoral, antimicrobial, and antilipidemic activities attributed to its polyphenolic
content. We studied the polyphenolic pattern of L. cuneifolia during different phenological stages:
flowering, fruiting, and post-fruiting. The highest total phenolic content was found in stems at postfruiting (214 ± 12.1 mg gallic acid eq·g−1 DW) and fruiting (209 ± 13.7 mg gallic acid eq·g−1 DW),
followed by post-fruiting leaves (207 ± 17.5 mg gallic acid eq·g−1 DW). Flavonoids accumulated
at higher levels in leaves and hydroxycinnamic acids in leaves at flowering and post-fruiting. The
polyphenolic pattern was similar between organs from wild plants and in vitro cultures, although
at a significantly lower level in the latter ones. The performance of calli growing under a 16 h
photoperiod in a modified White medium with 1-naphthalene acetic acid (2.50 µM) and Kinetin
(9.20 µM) was better than in the dark. When calli grew in media only with auxins (IAA, NAA, and
2,4-D, all at 2.50 µM concentration), its growth and polyphenolic content improved. Cell suspensions
with 2.50 µM NAA and 9.20 µM KIN grew slowly and produced very small amounts of polyphenols.
As for the antioxidant activity, it was detected in all samples (approximately 1000 µmol trolox
eq·g−1 DW) except fruits, where a lower value was found (328 µmol trolox eq·g−1 DW). In vitro
cultures have the lowest antioxidant activity when compared to methanolic extracts from organs
of wild specimens. Finally, antimutagenic or mutagenic activity in wild plants and in vitro culture
extracts was not detected by the Ames test.
creativecommons.org/licenses/by/
4.0/).
Plants 2021, 10, 1713. https://doi.org/10.3390/plants10081713
https://www.mdpi.com/journal/plants
Plants 2021, 10, 1713
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Keywords: antioxidant capacity; condensed tannins; dedifferentiated cultures; flavonoids; hemiparasite; hydroxycinnamic acids; mistletoe; phenolics; secondary metabolites
1. Introduction
Mistletoes are plant species from the Loranthaceae and Santalaceae families, spread
worldwide. Among the approximately 1000 Loranthaceae species, 300 are endemic to
America. The genus Ligaria is represented by L. teretifolia (Rizzini) Kuijt, endemic to Brazil,
and L. cuneifolia (Ruiz & Pav.) Tiegh, from Uruguay, Brazil, Perú, Chile, and Argentina [1,2].
L. cuneifolia is a hemiparasite species that, due to its morphological similarities and growth
behavior, was used as a substitute for the European mistletoe (Viscum album L., Santalaceae)
by the first European immigrants [3]. Ethnobotanical studies have reported L. cuneifolia
use as antihemorrhagic, abortive, emmenagogue, and oxytocic and against cephalgia,
gastralgias, sore throat, and hypothermia [2]. On the other hand, pharmacological studies
have demonstrated that L. cuneifolia extracts decreased cholesterol and lipid blood levels in
rats [4,5], had antitumoral activity [6], produced a reduction in cell proliferation in murine
lymphoma [7], had a bactericidal effect against phytopathogens and clinical pathogens [8],
and displayed a strong in vitro antioxidant activity [9–11]. Therefore, L. cuneifolia use
for the treatment of cardiovascular diseases and cancer is a promising alternative [12,13].
Phytochemical studies have identified several compounds potentially responsible for the
above-mentioned activities. The flavonol quercetin (QE) was identified in L. cuneifolia
specimens growing on different hosts and coming from different regions. QE could be
found free or as a 3-O-glycoside derivative with glucose, xylose, rhamnose, or arabinose.
Leucoanthocyanidins, catechin-4-ß-ol, and proanthocyanidins (PA) as polymers, oligomers,
and dimers that produced cyanidin after hydrolysis were also reported [14]. Dobrecky [15]
has also identified QE-3-O-(2′′ -O-galloyl) rhamnoside, QE-3-O-(3′′ -O-galloyl) rhamnoside,
QE-3-O-(2′′ galloyl)-arabinofuranoside, and QE-3-O-(2′′ -O-galloyl)-arabinopyranoside.
Production of secondary metabolites, including plant polyphenolics, depends on numerous
factors, such as growth conditions (light, temperature, altitude, nutrient availability),
plant phenological stage, and organ. Changes in the polyphenolic content are related to
variations in the expression of the genes encoding the activity of enzymes involved in
their biosynthesis [16]. Consequently, the analysis of polyphenolic compounds in different
phenological stages and organs is relevant to increase the knowledge on polyphenol
dynamics in L. cuneifolia and to harvest it at the highest levels of bioactive compounds.
In this work, we studied the dynamics of polyphenolic production in different organs of
L. cuneifolia wild specimens in different phenological stages.
On the other hand, the establishment of L. cuneifolia in vitro cultures as a source of
plant material appears as an environmentally friendly strategy to avoid the excessive
exploitation of the species. In addition, these cultures have the advantage of providing
plant material of uniform quality produced under controlled environmental conditions free
from pests and diseases. There are only a few reports about the establishment of in vitro
cultures from Loranthaceae species [17,18]. We have previously determined the conditions
to initiate in vitro calli cultures from L. cuneifolia on White medium with 500 mg L−1 casein
hydrolysate, 100 mg L−1 myo-inositol, B5 vitamins, 4% (w/v) sucrose, 2.50 µM NAA,
and 9.20 µM KIN as plant growth regulators (PGRs) and a 16 h photoperiod [19]. The
behavior of most plant cultures depends on the quality, intensity, and duration of the light
period, since the activity of many of the enzymes involved in the biosynthetic pathways
of metabolites is influenced by light [20]. Therefore, in the present work, we continued
the study of in vitro calli induction and growth behavior, analyzing the influence of two
different photoperiods. Once the calli are formed, it is necessary to improve their growth
and plant metabolite production. The influence of PGRs on the production of secondary
metabolites in in vitro cultures of different plant species is well known [21–23]. Accordingly,
in this study, we tested different PGRs on calli growth and polyphenol yield over time. In
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addition, we studied the influence of inoculum size on cell suspension culture initiation, a
key growth variable [21]. We measured their polyphenol production over time as well.
Finally, we tested the antioxidant, mutagenic, and antimutagenic activities of wild
specimens and in vitro culture extracts.
2. Results
2.1. Dynamics of Polyphenols in Different Organs and Phenological Stages of Wild Plants
2.1.1. Spectrophotometric Analysis
We found significant differences in the total phenolic content (TPC; p < 0.05) among
organs (leaves, stems, flowers, and fruits) in the three studied phenological stages (fruiting,
post-fruiting, and flowering). The highest values corresponded to stems at post-fruiting
(214 ± 12.1 mg GA eq·g−1 DW) and fruiting (209 ± 13.7 mg GA eq·g−1 DW), followed by
post-fruiting leaves (207 ± 17.5 mg gallic acid eq·g−1 DW). The lowest values were found
in fruits (81.7 ± 0.00 mg GA eq·g−1 DW; Tables 1 and 2).
Table 1. Concentration of total phenolics (mg gallic acid eq·g−1 DW) according to the phenological
stage and organ from Ligaria cuneifolia. Results were expressed as mean ± SD.
Phenological Stage
Organ
Total Phenolics (mg Gallic Acid eq·g−1 DW) 1
Flowering
Flowers
Leaves
Stems
179 ± 22.0
168 ± 1.9
183 ± 13.9
Fruiting
Fruits
Leaves
Stems
81.7 ± 0.00
170 ± 4.86
209 ± 13.7
Post-fruiting
Leaves
Stems
207 ± 17.5
214 ± 12.1
1
Two-way ANOVA results for phenological stage: F (2.24) = 15.52, p < 0.001; and for organ: F (1.24) = 15.94,
p < 0.001.
Table 2. Tukey’s post hoc test comparing Ligaria cuneifolia leaves and stems in different phenological stages for the variable
total phenolics. df: degrees of freedom; SE: standard error.
Organ
Phenological Stage
Organ
Phenological Stage
Mean Difference
SE
df
t
ptukey
Leaves
Flowering
Leaves
Leaves
Stems
Stems
Stems
Fruiting
Post-fruiting
Flowering
Fruiting
Post-fruiting
−2.45
−39.58
−15.29
−40.87
−45.94
8.74
9.09
9.58
8.74
9.09
24
24
24
24
24
−0.28
−4.355
−1.596
−4.674
−5.055
1
0.003 1
0.609
0.001 1
<0.001 1
Fruiting
Leaves
Stems
Stems
Stems
Post-fruiting
Flowering
Fruiting
Post-fruiting
−37.13
−12.84
−38.42
−43.49
8.20
8.74
7.82
8.20
24
24
24
24
−4.527
−1.468
−4.912
−5.302
0.002 1
0.686
<0.001 1
<0.001 1
Post-fruiting
Stems
Stems
Stems
Flowering
Fruiting
Post-fruiting
24.29
−1.29
−6.36
9.09
8.20
8.57
24
24
24
2.673
−0.157
−0.742
0.118
1
0.974
Flowering
Stems
Stems
Fruiting
Post-fruiting
−25.58
−30.65
8.74
9.09
24
24
−2.926
−3.373
0.071
0.027 1
Fruiting
Stems
Post-fruiting
−5.07
8.20
24
−0.618
0.989
Stems
1
Significant differences (p < 0.05).
As for flavonoids (FL), the highest content was found in leaves in the three analyzed
phenological stages (Figure 1).
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Regarding hydroxycinnamic acids (HCA), significant differences (p < 0.05) were found
between leaves and stems; however, no significant differences (p > 0.05) were found among
phenological stages. The highest values corresponded to leaves harvested at the flowering
(3.02 ± 0.62 mg CA eq·g−1 DW) and post-fruiting stages (3.06 ± 0.9 mg CA eq·g−1 DW;
Table 3).
Figure 1. Total flavonoid content (mg quercetin eq·g−−1 DW) measured by UV spectrophotometry in Ligaria cuneifolia’s
methanolic extracts of leaves, stems, flowers, and fruits, harvested in different phenological stages (flowering, fruiting, and
post-fruiting).
Table 3. Concentration of total hydroxycinnamic acids (mg chlorogenic acid eq·g−1 DW) according
to the phenological stage and organ from Ligaria cuneifolia. Results were expressed as mean ± SD.
−
−
Phenological Stage 1
Organ 1
Hydroxycinnamic Acids
(mg Chlorogenic Acid eq·g−1 DW)
Flowering
Flowers
Leaves
Stems
−
2.05 ± 0.40
3.02 ± 0.62
2.50 ± 0.26
Fruiting
Fruits
Leaves
Stems
2.77 ± 0.01
2.42 ± 0.28
2.24 ± 0.41
Post-fruiting
Leaves
Stems
3.06 ± 0.69
2.40 ± 0.49
−
1
Two-way ANOVA results for phenological stage: F (1.24) = 6.601, p = 0.017 and for organ F (2.24) = 2.700,
p = 0.088.
No significant differences in proanthocyanidin (PA) content were found among the
different organs and phenological stages (p > 0.05; Table 4).
Table 4. Total proanthocyanidin content in different Ligaria cuneifolia organs and phenological stages.
Results were expressed as mean ± SE.
1
Phenological Stage 1
Organ 1
Proanthocyanidins
(mg Catechin eq·g−1 DW)
Flowering
Flowers
Leaves
Stems
32.5 ± 9.60
32.4 ± 7.25
32.4 ± 5.99
Fruiting
Fruits
Leaves
Stems
23.9 ± 5.0
29.0 ± 12.0
32.7 ± 6.87
Post-fruiting
Leaves
Stems
35.3 ± 5.79
34.6 ± 8.59
−
Two-way ANOVA results for phenological stage: F (1.24) = 0.103, p = 0.751 and for organ F (2.24) = 0.656,
p = 0.528.
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2.1.2. Thin-Layer Chromatography (TLC) Analysis
The TLC analysis showed that the pattern of FL and HCA was similar in all organs
and phenological stages studied (Figure 2a). As for their content, the highest polyphenolic
values corresponded to the post-fruiting stage. The PA (+) catechin was detected in all
organs and phenological stages studied (Figure 2c). This finding was confirmed by bidimensional TLC where a similar pattern of flavonoids, HCA, and PA was found in leaves
and stems harvested in the post-fruiting stage (Figure 2b,d).
Figure 2. Mono-dimensional (a,c) and bi-dimensional (b,d) analysis of methanolic extracts from
Ligaria cuneifolia organs harvested in different phenological stages. (a,c) Methanolic extracts of L:
leaves; S: stems; FR: fruits; F: flowers; standards: R (rutin) and C (catechin); (b,d) methanolic extracts
of leaves and stems from wild specimens harvested in the post-fruiting stage (equal parts mix). Spray
reagents: (a,b) AEDBE: 2-aminoethyl diphenyl borate ester, Sigma-Aldrich. Wavelength: 366 nm;
(c,d) ethanolic solution of 5% (v/v) vanillin/hydrochloric acid.
2.1.3. HPLC-UV Analysis
Catechin was the main compound found in wild plants. QE glycosides (QE-3-Oglycoside, QE-3-O-xyloside, QE-3-O-arabinopyranoside, QE-3-O-arabinofuranoside, QE3-O-rhamnoside, QE-3-O-2-galloyl-arabinofuranoside, QE-3-O-2-galloyl-rhamnoside and
QE-3-O-3-galloyl-rhamnoside) were found at lower levels (Figure 3, Table 5). The highest
−
content of total metabolites was found in the extracts from stems (20.44 ± 0.36 mg
g−1
−
1
DW), followed by leaves (19.64 ± 0.24− mg g DW) in the post-fruiting stage. The lowest
−1 DW).
values corresponded to fruits (4.32 ± 0.04
− mg g
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Figure 3. Chromatogram resulting from the analysis by HPLC-UV of the methanolic extract of Ligaria cuneifolia stems
from specimens harvested in the post-fruiting stage. C (Catechin), Q-3-O-gluc (Q-3-O-glucoside), Q-3-O-xyl (Q-3-Oxyloside), Q-3-O-AP (Q-3-O-arabinopyranoside), Q-3-O-AF (Q-3-O-arabinofuranoside), Q-3-O-2-gal-AF (Q-3-O-2-galloylarabinofuranoside), Q-3-O-R (Q-3-O-rhamnoside), Q-3-O-2-gal-R (Q-3-O-2-galloyl-rhamnoside) and Q-3-O-3-gal-R (Q-3-O3-galloyl-rhamnoside).
2.1.4. Histochemical Analysis
Histochemical analysis showed that flavonoids accumulated in the epidermis and in
the first parenchyma layers of leaves, and with less intensity in the deeper layers of tissue
(Figure 4c,d). PA were found in the epidermis, the assimilating parenchyma and vaguely in
the central spongy parenchyma (Figure 4a,b). In the primary stems, FL accumulated mainly
in the epidermis and first layers of parenchyma (Figure 4g,h), PA in the epidermis, the
primary cortex and at lower concentration in the stem pith (Figure 4e,y,f). In the embryo,
FL were found in the external tissues (Figure 5k,l), and PA in the epidermis and at a high
concentration in parenchyma (Figure 4i,j).
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Table 5. Concentration of polyphenols detected by HPLC-UV of methanolic extracts from different organs (leaves, stems, flowers, and fruits) harvested in different phenological
stages (flowering, fruiting, and post-fruiting), from calli grown with different PGRs (2,4-D, NAA and IAA all at 2.5 µM) and grown under different illumination conditions (16 h
photoperiod and darkness) in CWM with 2.50 µM NAA and 9.20 µM KIN. C (Catechin). Q-3-O-gluc (Q-3-O-Glucoside). Q-3-O-xyl (Q-3-O-xyloside). Q-3-O-AP (Q-3-O-arabinopyranoside).
Q-3-O-AF (Q-3-O-arabinofuranoside). Q-3-O-2-gal-AF (Q-3-O-2-galloyl-arabinofuranoside). Q-3-O-R (Q-3-O-rhamnoside). Q-3-O-2-gal-R (Q-3-O-2-galloyl-rhamnoside) and Q-3-O-3-gal-R
(Q-3-O-3-galloyl-rhamnoside). ND: not detected. Values are expressed as mean ± SEM.
Concentration (mg g−1 Dry Weight)
Phenological
Stage/Treatment
Organ
C
Q-3-O-gluc
Q-3-O-xil
Q-3-O-AP
Q-3-O-AF
Q-3-O-2-gal-AF
Q-3-O-R
Q-3-O-2-gal-R
Q-3-O-3-gal-R
Total
Flowering
Flowering
Flowering
Flowers
Stems
Leaves
8.65 *a ± 0.08
7.43 ± 0.01
7.41 ± 0.31
0.16 ± 0.02
0.18 ± 0.02
0.32 *a ± 0.18
0.30 *a ± 0.02
0.24 *b ± 0.04
0.56 *c ± 0.01
0.35 ± 0.15
0.33 ± 0.05
0.66 *a ± 0.08
0.63 *a ± 0.05
0.35 *b ± 0.03
1.01 *c ± 0.13
0.48 *a ± 0.06
0.33 *b ± 0.03
0.82 *c ± 0.06
0.83 *a ± 0.15
0.49 *b ± 0.03
1.42 *c ± 0.58
0.32 *a ± 0.06
0.41 *b ± 0.09
0.73 *c ± 0.03
0.55 *a ± 0.15
0.25 *b ± 0.01
0.74 *c ± 0.10
12.27 *a ± 0.63
10.01 *b ± 0.19
13.71 *c ± 0.69
Fruiting
Fruiting
Fruiting
Stems
Leaves
Fruit
6.96 *a ± 0.84
8.66 *b ± 0.66
0.86 *c ± 0.02
0.18 ± 0.02
0.25 *a ± 0.01
0.20 ± 0.01
0.26 ± 0.06
0.32 *a ± 0.04
0.24 ± 0.02
0.28 *a ± 0.08
0.22 *b ± 0.04
0.50 *c ± 0.02
0.42 *a ± 0.12
0.56 *b ± 0.08
0.72 *c ± 0.01
0.37 *a ± 0.03
0.51 *b ± 0.07
0.72 *c ± 0.01
0.67 *a ± 0.29
0.91 *b ± 0.29
0.40 *c ± 0.02
0.50 *a ± 0.06
0.62 *b ± 0.04
0.16 *c ± 0.01
0.47 *a ± 0.05
0.81 *b ± 0.01
0.52 *c ± 0.02
10.11 *a ± 1.15
12.86 *b ± 0.30
4.32 *c ± 0.04
Post-fruiting
Post-fruiting
Stems
Leaves
13.81 ± 0.98
12.16 ± 0.95
0.28 ± 0.01
0.24 ± 0.02
0.57 ± 0.05
0.89 ± 0.15
0.63 ± 0.03
0.79 ± 0.13
0.99 ± 0.09
1.24 ± 0.08
0.98 ± 0.12
1.27 ± 0.11
1.02 ± 0.20
1.65 ± 0.03
0.54 ± 0.01
0.53 ± 0.11
1.62 ± 0.97
0.87 ± 0.13
20.44 ± 0.36
19.64 ± 0.24
2.4-D 2.5 µM
IAA 2.5 µM
NAA 2.5 µM
Calli
Calli
Calli
0.99 *a ± 0.51
1.97 *b ± 0.31
0.60 *c ± 0.36
0.08 *a ± 0.01
0.11 *b ± 0.01
0.02 *c ± 0.02
0.08 *a ± 0.02
0.12 *b ± 0.04
0.04 *c ± 0.02
0.15 *a ± 0.01
0.27 *b ± 0.07
0.08 *c ± 0.01
0.19 ± 0.05
0.21 *a ± 0.05
0.09 *b ± 0.05
0.13 *a ± 0.05
0.10 *b ± 0.04
0.04 *c ± 0.02
0.31 *a ± 0.25
0.37 *b ± 0.29
0.10 *c ± 0.08
0.10 *a ± 0.08
0.05 *b ± 0.01
0.02 *c ± 0.01
0.05 *a ± 0.03
0.07 *b ± 0.03
0.01 *c ± 0.01
2.08 *a ± 0.02
3.27 *b ± 0.77
1.00 *c ± 0.48
Darkness
Light
Calli
Calli
0.07 ± 0.01
0.02 ± 0.01
ND
0.03 ± 0.01
ND
0.07 ± 0.03
ND
0.23 ± 0.05
0.09 ± 0.01
0.19 ± 0.09
0.07 ± 0.03
0.08 ± 0.05
0.07 ± 0.03
0.19 ± 0.08
0.04 ± 0.01
0.03 ± 0.01
0.07 ± 0.01
0.03 ± 0.01
0.40 ± 0.02
0.87 ± 0.08
Each analyte was compared among organs of the same phenological stage or PGR. Mean values followed by an asterisk and different letters (a–c) represent statistically significant differences (p < 0.05, Tukey’s
multiple comparison test).
Plants 2021, 10, 1713
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Figure 4. Cross sections of Ligaria cuneifolia leaf (a–d), stem (e–h) and embryo (i–l). Subfigures (a,e,i) correspond to cross
sections of the three organs prior to reaction with vanillin–hydrochloric acid, observed under a bright-field microscope; the
subfigures (b,f,j) correspond to the same sections, but after the reaction with the vanillin–hydrochloric reagent in which
a characteristic red coloration typical of proanthocyanidins is observed. Subfigures (c,g,k) are cross sections of the three
organs prior to reaction with AEDBE observed under a fluorescence microscope; subfigures (d,h,i) correspond to the same
sections, but after reaction with AEDBE in which the presence of flavonoids is revealed by a characteristic yellow coloration.
2.2. In Vitro Culture Initiation, Growth Kinetics, and Polyphenolic Content under Different
Culture Conditions
2.2.1. Light Influence on Calli Induction, Growth and Polyphenolic Content
Calli induction rate was 84.65 ± 9.23% in darkness, while it was 71.19 ± 3.70% with a
16 h photoperiod. No correlation between both variables was demonstrated by chi-square
and Fisher’s tests (p > 0.05). However, the calli morphology differed, being green and
compact under light, or whitish and friable in the dark (Figure 5). The kinetic parameters
corresponding to calli growing under illumination were GI = 1.17, µ = 0.01, and dt = 62.7
(Figure 6). Maximal biomass (524 ± 40 mg FW) was achieved at week
μ 14. The cell growth
curve did not display a lag phase, showed a very slow exponential phase, and entered
the stationary phase by week 14. On the other hand, after one week in darkness calli
arrested their growth (maximal biomass: 346 ± 10 mg FW) and by the 3rd week entered
the death phase.
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Figure 5. Aspect of calli induced from embryos under continuous darkness (a,c,e) and under a 16 h light photoperiod (b,d).
μμ
Note that calli were formed on the extreme of the hypocotyl in all cases (a–c). Culture media: CWM with 2.5 µM NAA:
μμ
9.2 µM KIN.
Figure 6. Growth kinetics of Ligaria cuneifolia calli initiated under a 16 h photoperiod (a) and in darkness (b), expressed as
fresh biomass (g) over time (weeks). Calli were grown in CWM with 2.5 µM
μμ NAA and 9.2 µM
μμ KIN.
As for TPC, FL, and HCA contents, significant differences (p < 0.001) were found
between cultures maintained under illumination or in darkness. Maximum yields for calli
grown under light were 7.84 ± 0.01 mg GA eq− ·g−1 DW at 4th week, 1.86 ± 0.16 mg QE
−−
−
eq·g−1 DW and 0.26 ± 0.07 mg −CA eq·g−1 DW at the 2nd week of culture, respectively.
−
For calli grown in darkness, maximum yields were 6.62 ± 0.01 mg− GA eq·g−1 DW and
−
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−
−
1.11 ± 0.01 mg QE eq·g−1 DW at the 2nd week, and 0.12 ± 0.06 mg CA eq·g−1 DW at the
1st week of culture, respectively. PA was not detected by spectrophotometry in any case.
On the other hand, TLC revealed more intense spots in extracts from calli grown under
16 h photoperiod than in darkness. The PA(+) catechin was only detected by this method
(Figure 7b,d,e,h,i). HPLC-UV analysis showed that the total amount of polyphenols in
−
−
calli was 2-fold higher in light (0.87 ± 0.08 mg g−1 ) than in darkness (0.40 ± 0.02 mg g−1 )
methanolic extracts (Table 5).
Figure 7. Mono-dimensional (a,b,f) and bi-dimensional (c–e,g–i) TLC analysis of methanolic extracts of Ligaria cuneifolia
in vitro cultures. (a,f) Calli grown in CWM with different PGRs (IAA, NAA, and 2,4-D) at a concentration of 2.5 µM; μ
(b) Cell
μ NAA and
suspensions (SUSP), calli grown under 16 h photoperiod (CL) and in darkness (CD) in CWM with 2.50 µM
μ KIN; (c,g) calli grown in White medium with 2,5 µM
μ NAA harvested at 6th week of culture; (d,h) calli grown in
9.20 µM
CWM under 16 h photoperiod harvested at the 2nd week of culture; (e,i) calli grown in CWM under continuous darkness
harvested at the 2nd week of culture. Spray reagents: (a–e): AEDBE: 2-aminoethyl diphenyl borate ester, Sigma-Aldrich,
wavelength: 366 nm; (f–i): ethanolic solution of 5% (v/v) vanillin/hydrochloric acid. 2,4-D: 2,4-dichlorophenoxyacetic acid;
IAA: indoleacetic acid; NAA: 1-naphthaleneacetic acid; C: catechin.
2.2.2. Influence of PGRs and Inoculum Size on Growth Kinetics and Polyphenolic Content
All PGRs at the lowest concentration (2.5
μ µM) with inoculum sizes between 250
and 500 mg FW per tube were effective in inducing growth (Figure 8). On the other
hand, none of them were effective at the higher concentration of auxinsμ(5 and 10 µM) or
inoculum sizes lower than 250 mg FW per tube. The cell growth curves showed that the
performance of calli growing in culture media with IAA and 2,4-D as PGR was better than
with NAA. No significant differences were observed between the GI and growth specific
μ
rate (µ) from cultures growing in media with IAA (1.60 and
0.02 d−1 , respectively) or 2,4-D
−
(1.57 and
0.02 d−1 , respectively), whileμ GI and µ were lower with NAA (1.28 and− 0.01 d−1 ,
−
respectively). The doubling times (dt ) of calli in media with 2.5 µM 2,4-D, IAA, or NAA
μ
as PGR were 30.27 d, 42.27 d and 68.35 d, and the maximal biomass was 513 ± 73 mg
FW at the 7th week, 455 ± 49 mg FW at the 8th week, and 453 ± 47 mg FW at the 6th
week, respectively.
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0.550
0.500
Fresh weight (g)
0.450
0.400
2,4-D
0.350
IAA
0.300
NAA
0.250
0.200
0.150
0
1
2
3
4
5
6
7
8
Time (weeks)
Figure 8. Growth kinetics of Ligaria cuneifolia calli grown in a semi-solid medium (CWM), with
different auxins (IAA, NAA or 2,4-D at 2.5μµM), initiated from “high” inocula (250 to 500 mg FW).
Photoperiod 16 h, temperature 24 ± 2 ◦ C.
The spectrophotometric analysis has determined that the higher TPC corresponded
to treatments with IAA and NAA. As for FL, the higher amounts corresponded to calli
growing in media with IAA and 2,4-D. No significant differences were found in HCA
content among treatments. Finally, the highest amount of PA corresponded to calli growing
in the presence of IAA and NAA (Figure 9).
Mono-dimensional and bi-dimensional TLC confirmed that calli have the same pattern
of polyphenolic compounds (Figure 7a,c) as the plant but at a lower concentration. The
PA(+) catechin was detected (Figure 7f,g). HPLC-UV analysis (Figure 10) showed that, as
in the adult plant, catechin was the main compound in calli, but the amounts were 10- to
20-fold lower (Table 5).
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Figure 9. Polyphenolic content of methanolic extracts of calli grown in CWM with different auxins at
μ Results are presented as the mean with standard error bars. (A) Total phenolics expressed as
2.5 µM.
−
−
mg GA eq·g−1 DW; (B) flavonoids expressed as mg QE eq·g−1 DW; (C) total hydroxycinnamic acids
−
−
expressed as mg CA eq·g−1 DW; (D) total proanthocyanidins expressed as mg C eq·g−1 DW. 2,4-D:
2,4-dichlorophenoxyacetic acid; IAA: indoleacetic acid; NAA: naphthaleneacetic acid; GA: gallic acid;
QE: quercetin; CA: chlorogenic acid; C: catechin; DW: dry weight. Letters above the bars indicate
same groups (p < 0.05) according to Tukey’s test (plots A,D) and Games–Howell test (plot B). In plot
C, non-significant differences were detected by ANOVA (p = 0.174).
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Figure 10. Chromatogram resulting from the HPLC-UV analysis of the methanolic extract of Ligaria cuneifolia calli grown
μ
in a semi-solid medium (CWM plus 2.5 µM IAA) and harvested at week 6. C (Catechin), Q-3-O-gluc (Q-3-O-glucoside),
Q-3-O-xyl (Q-3-O-xyloside), Q-3-O-AP (Q-3-O-arabinopyranoside), Q-3-O-AF (Q- 3-O-arabinofuranoside), Q-3-O-2-galAF (Q-3-O-2-galloyl-arabinofuranoside), Q-3-O-R (Q-3-O-rhamnoside), Q-3-O-2-gal-R (Q-3-O-2-galloyl-rhamnoside) and
Q-3-O-3-gal-R (Q-3-O-3-galloyl-rhamnoside).
2.2.3. Establishment of Cell Suspension Cultures. Growth Kinetics and
Polyphenolic Content
−
A low inoculum size (3 mg mL−1 ) was not
effective in inducing growth. On the
−
other hand, with a higher inoculum size (25 mg mL−1 ) the growth curve from cell suspensions was sigmoidal with a lag phase of 7 days, followed by an exponential phase that
extended up to the 28th day, when the
culture entered the stationary phase (Figure 11).
−
μ
The kinetic parameters
were
GI
=
1.07,
µ
= 0.01 d−1 , and dt = 66.89 d, with a biomass yield
−
−
1
of 30.33 ± 0.67 mg mL . Microscopic analysis of the suspensions showed the presence
of single or grouped cells forming aggregates. Cells had different morphologies, from
spherical to oblong. The presence of starch was also observed (positive Lugol’s test and
confirmed with polarized light). Evans blue and FDA tests proved that all cultures were
viable (Figure 12). Mono-dimensional TLC showed a profile similar to that of calli and
wild plant extracts but at a lower intensity (Figure 7b). The concentration of TPC, HCA,
and PA was higher in the first week of culture and fell abruptly as the culture entered the
exponential phase of growth. On the other hand, the low FL content fell smoothly from
−
−
the 7th day of culture (from 0.35 ± 0.04 mg QE eq·g−1 DW to 0.24 ± 0.04 mg QE eq·g−1
DW; Figure 11). As was observed in calli, cell suspension cultures grew poorly under the
conditions tested and did not produce a significant amount of polyphenolic compounds.
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Figure 11. Growth and polyphenolic content in Ligaria cuneifolia cell suspension cultures growing
μ µM NAA and 9.20
μ µM KIN as PGR. Inoculum size: 25 mg −mL−1 , photoperiod:
in CWM with 2.50
−
μ
μ
−
eq·g−1 DW) and proantho16 h, time of culture: 35 days (a,b). (a) Total phenolics (mg gallic acid
−
−
−
cyanidins content (mg catechin
eq·g−1 DW); (b) total flavonoids (mg quercetin eq
·g−1 DW) and
−
−
−
−
1
hydroxycinnamic acids content (mg chlorogenic acid eq·−g DW).
Figure 12. Cell suspension cultures of L. cuneifolia observed under the bright-field microscope. (a) Cell
aggregates (40×); note the presence of intracellular starch granules; (b) cells observed under the
bright-field microscope; positive Lugol’s test confirmed the presence of starch (40×); (c) fluorescent
viable cell by the transformation of DAF into fluorescein (observation under UV light, 40×); a and b
had the ability to exclude Evans blue at the plasma membrane and maintain their natural color.
2.3. Antioxidant Activity of Wild Plants and In Vitro Cultures
All organs at μ
the phenological− stages tested showed similar antioxidant activity (ap−−1 DW) except in the case of fruits that showed a lower
μ
proximatelyμ1000 µmol
trolox
− eq·g
−
−
1
μ
value (328 µmol
trolox eq·g DW; Figure 13). As for in vitro cultures, both calli and
cell suspension cultures showed antioxidant activity, but it was lower than inμplants. On
μ
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−
−
μ under illumination
the other hand, antioxidant activity was higher
(28.6 ± 8.21 µmol
−1 DW) thanμin darkness (23 ± 7.54 µmol trolox eq·g−1 DW) with the PGR
·
g
trolox
eq
−
−
μ
μ
−
μ
μ with auxins, the
In calli cultures growing
highest
ratio 2.50 µM μNAA and 9.20 µM KIN.
−
value was obtained in CWM with 2.5 µM IAA as PGR (137 ± 7.92 −µmolμtrolox eq·g−1
μ
μ
−
μ
DW). As for cell suspension cultures
(25 mg mL−1 inoculum
size, CWM
with 2.50 µM
−
μ
μ
NAA and 9.20 µM KIN), their antioxidant activity was 44.1 ± 9.84
µmol trolox eq·g−1 DW
−
μ
(Figure 14).
μ
−
Figure 13. Antioxidant
activity expressed as trolox equivalent antioxidant capacity (µmol trolox
eq·g−1 DW), in organs from L. cuneifolia harvested in different phenological stages. μ
−
μ
μ
Figure 14. Antioxidant activity expressed as trolox equivalent antioxidant
capacity
(µmol trolox
−
μ
eq·g−1 DW), from in vitro cultures of L. cuneifolia growing in CWM under different conditions:
μ
μ 9.20 µM μ
suspension cultures in darkness with 2.50 µM NAA and
KIN;− calli growing under
a 16 h
μ
−
μ under a 16 h
μ 9.20 µM KIN; or calli growing
photoperiod or darkness with 2.50 µM NAA and
μ
μ
−
1
photoperiod with IAA, NAA or 2,4-D at 2.5 µM. For calli culture 6.5 g L of plant agar was added
−
μ
to CWM.
2.4. Mutagenicity and Antimutagenicity Assays of Wild Plants and In Vitro Culture Extracts
2.4.1. Mutagenicity Assay
As can be seen in Tables 6 and 7, none of the extracts or dilutions assayed showed
mutagenic activity on S. typhimurium strains TA 98 and TA 100, with or without metabolic
activation (RC values less than 2 indicate absence of mutagenicity). The results of the
positive controls (not shown in the tables and not included in the CR calculations) for both
strains gave values of CFU (colony forming units) greater than 1000 without metabolic
μ
μ
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activation, while when metabolic activation was performed, CFU values were 704.7 ± 55
for TA 100 and 919.67 ± 43.02 for TA 98.
Table 6. Results of the Ames test in TA 100 and TA 98 S. typhimurium strains with (+S9) and without
(−S9) metabolic activation. A mixture of equal parts of leaf and stem methanolic extracts from
specimens harvested in the post-fruiting season was tested. Results were expressed as the reversion
coefficient (RC). SD: standard deviation.
RC ± SD—Adult Specimen Extracts
1
4
1
2
diluted extract
diluted extract
Direct extract
TA 100 + S9
TA 100 − S9
T 98 + S9
TA 98 − S9
0.95 ± 0.09
0.97 ± 0.04
0.84 ± 0.04
1.02 ± 0.05
1.01 ± 0.03
0.76 ± 0.17
1.01 ± 0.12
0.99 ± 0.14
0.78 ± 0.06
1.00 ± 0.05
0.96 ± 0.09
0.86 ± 0.18
Table 7. Results of the Ames test in TA 100 and TA 98 S. typhimurium strains with (+S9) and without
(−S9) metabolic activation. Methanolic extracts from calli grown in White medium with 500 mg L−1
casein hydrolysate, 4% (w/v) sucrose, and 2.50 µM NAA were tested. Results were expressed as the
reversion coefficient (RC). SD: standard deviation.
RC ± SD—In Vitro Cultures Extracts
1
4
1
2
diluted extract
diluted extract
Direct extract
TA 100 + S9
TA 100 − S9
T 98 + S9
TA 98 − S9
1.00 ± 0.07
1.03 ± 0.03
0.92 ± 0.07
1.02 ± 0.06
1.03 ± 0.04
0.94 ± 0.04
1.07 ± 0.04
1.02 ± 0.12
1.00 ± 0.03
1.04 ± 0.05
1.01 ± 0.13
0.81 ± 0.18
2.4.2. Antimutagenicity Assay
Once the assumption of homogeneity of variances was confirmed by the Bartlett test
(p > 0.05), the results were compared using the Kruskal–Wallis test, and no significant
differences were observed (p > 0.05) between the positive controls and the extracts tested.
This indicates that the extracts obtained from wild specimens of L. cuneifolia did not show
an antimutagenic effect.
3. Discussion
Phenolic compounds are redox-active species widely distributed in the plant kingdom.
Among them, we focused our analysis on flavonoids (FL), hydroxycinnamic acids (HCA),
and proanthocyanidins (PA) that, besides lectins, betulin, and betulinic acid, were the major
components found in L. cuneifolia and considered responsible for their pharmacological
activities [24]. Although phenolic compounds are synthesized in all parts of the plant, their
content varies during plant growth and development. The content of phenolic compounds
may vary depending on biotic (bacteria, fungi, parasites, predators) and abiotic (water,
light, salts, chemicals, temperature, humidity, geographical variations, etc.) factors, the
growth stage, and the part of the plant [25,26]. In the case of hemiparasites, another variable
is added: the host. In this study, specimens were collected in Villa de Merlo (32◦ 21′ 22.5′′ S,
65◦ 00′ 20.5′′ W, 796 m.a.s.l.), in the province of San Luis. This location belongs to the Cuyo
geographical region, an arid or semiarid climate with an average annual precipitation of
about 100 to 500 mm and a pronounced temperature range from extremely hot temperatures
during the day, followed by cold nights. From a phytogeographical perspective, this zone
is located in the Neotropical region, specifically the Chaqueño domain with polymorphic
vegetation and varied weather, in which the continental type is predominant with moderate
to scarce rainfall, mild winters and warm summers [27]. Our research was focused on a
single host, Vachellia caven (Mol.) Mol (Fabaceae), a common host of L. cuneifolia. Previous
reports from our group also evaluated specimens from Barreal (31◦ 38′ 00′′ S, 69◦ 28′ 00′′ W,
1478 m.a.s.l.), in the province of San Juan, which is also part of the Cuyo geographical
region, the Neotropical phytogeographical region and the Chaqueño domain. In that study,
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samples growing on Prosopis chilensis (Molina) Stuntz, Prosopis flexuosa D.C., and Geoffroea
decorticans (Gillies ex Hook. & Arn.) Burkart, also from the Fabaceae family, were collected
during the post-bloom stage. For comparative purposes, specimens from the Catamarca
province (Belen, Puerta de San Jose, 27◦ 33′ 0′′ S, 67◦ 1′ 0′′ W, 1450 m.a.s.l.) developing on
different hosts (Olea europaea L. Oleaceae, Bulnesia retama (Gillies ex Hook. & Arn.) Griseb.
(Zygophyllaceae), Geoffroea decorticans (Gillies ex Hook. & Arn.) Burkart, and Prosopis
flexuosa D.C.) were also included in our prior study [15]. This area belongs to the Northwest
region, namely the Puna and is dry with a great temperature oscillation and mostly cold
with subzero temperatures at night. It has been suggested that the phytochemical profile
of mistletoes depends on the host of this parasitic plant [26]. However, the analysis of
these combined findings strongly suggests that, from a qualitative perspective, L. cuneifolia
FL fingerprint is highly conserved among geographical regions, climate conditions, and
host families.
With regard to polyphenolic content, stems and leaves displayed the highest values,
especially in the fruiting and post-fruiting stage. From a climatological point of view,
temperature variation is not significant but the average precipitation is at its peak, so
this period could be considered the “humid season.” This particular set of conditions
favors plant metabolism, which results in a polyphenolic increase. Aerial parts are largely
exposed to environmental conditions and different stressors, so a rapid turnover is generally
observed. HCAs are prevalent in leaves, followed by stems, and are not significantly
affected by the phenological stage. Polyphenols, and particularly FL, are secondary or
“specialized” metabolites that provide protection and are involved in defense mechanisms.
In terms of concentration, leaves showed the highest levels, especially at the post-fruiting
stage where catechin is the predominant compound. This is consistent with the role of
tannins as defense compounds. QE-3-O-glycosides and galloyl glycosides were also present
at lower values. Our results are in agreement with those found in V. album, in which leaf
extracts showed higher concentrations of total phenolics and flavonoids compared to fruit
and seed extracts [28]. Similarities in polyphenolic profile but quantitative differences
among growing seasons were also reported in C. palirus leaves. Several authors attributed
the increase in phenolics content to higher intensity of solar radiation [29–31]. There has
also been reported an increased expression of genes encoding phenylalanine ammonium
lyase, chalcone synthase, and flavanone-3-β-hydroxylase in leaves of Vaccinium myrtillus
exposed to sunlight [32]. These results correlate with those found in L. cuneifolia where the
post-fruiting stage showed the highest polyphenolic values, coinciding with the summer
season when solar radiation reaches its maximum. HCA were higher in leaves, and
were not affected by the phenological stage. These results correlate with those found in
Geoffroea decorticans extracts, in which leaves showed higher levels of HCA derivatives when
compared to stems [33]. Chapel [16], working with Calluna vulgaris (L.) Hull, reported that
the higher amounts of polyphenolic compounds, including HCA, were found in stems and
leaves at all phenological stages except during flowering, while no significant differences
in PA content were observed among different organs and phenological stages. As in
L. cuneifolia, PA were also found in adult plants of the Mexican mistletoes Phoradendron
bollanum and Viscum album subsp. austriacum [34]. HPLC results were consistent with a
previous spectrophotometric analysis; leaves and stems showed the highest levels of total
polyphenolics, especially in the post-fruiting stage, where catechin was the predominant
compound. QE-3-O-glycosides and galloyl glycosides were also present but at lower values.
A selective spatial distribution was also seen in histochemical analysis, where flavonols
and PA were located near the leaf surface, mostly in the epidermis and first parenchyma
layers, which is consistent with the results reported in other plant species. Positive results were obtained in the reaction with vanillin/HCl, in Microlaena stipoides, Eurycoma
longifolia, and Themeda triandra, which suggests the presence of condensed tannins and
their precursors. The resulting pink coloration could indicate the existence of flavan-4-ols
that produce anthocyanidins in the presence of concentrated HCl [35]. Ellis [36] found
tannin-like compounds in the epidermal cells of the leaves of 39 genera and 101 species of
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South African grasses after histochemical analysis. On the other hand, FL were detected by
histochemistry and confirmed by TLC, HPLC, UV/Vis spectroscopy, and mass spectrometry analysis, in Arabidopsis thaliana seedlings in three main zones: the cotyledonary node,
the hypocotyl, and the root apical end [37], which is in agreement with our results with
L. cuneifolia embryos.
As far as we know, there are few reports about the establishment of in vitro cultures
from hemiparasitic plants for plant secondary metabolite production, e.g., chlorogenic acid
production by Viscum album calli [38]. Most of the literature refers to the establishment of
in vitro cultures from hemiparasite species, and particularly in the Loranthaceae family,
to induce organogenesis [17,39,40], while phytochemical analysis was only performed in
plants [17,41,42]. To our knowledge, this is the first report on species from the Loranthaceae
family aiming at establishing in vitro culture conditions to produce polyphenolic compounds, particularly FL, HCA, and PA. Our results showed that L. cuneifolia calli presented
a similar pattern of polyphenolic compounds as the adult plant regardless of the tested
conditions. Illumination (16 h photoperiod) seemed to be a fundamental requisite for
long-term maintenance and to polyphenolic production of calli cultures in L. cuneifolia. The
positive effect of light on polyphenolic compounds accumulation could be related to the induction of some enzymes that participate in their biosynthetic pathway [43]. Shipilova [44],
working with tea-plant calli, reported the increased PAL activity in those growing under a
16 h photoperiod, with the consequent increase in flavan concentration. Several authors
have also reported the positive effect of light on growth and polyphenolic compound
production by in vitro cell cultures, such as López-Laredo [45] working with Tecoma stans
and Kumar [46] working with Basella rubra.
Considering the modest performance of calli in the previous assays and as PGRs are
culture media components with utmost relevance in in vitro culture growth and biosynthetic capacity, we tested the influence on growth and polyphenolic content of calli growing
on media with different auxins (IAA, NAA, and 2,4-D) at three concentrations (2.5, 5.0,
and 10.0 µM). IAA (2.5 µM) appeared as the most favorable plant growth regulator for
producing polyphenolic compounds. However, their amounts were low in comparison
with those from organs of wild plants, except for PA. This group of polyphenols is rarely
studied for in vitro production by plant cells [47–49].
As for cell suspension cultures, the presence of starch in cells was previously reported for calli cultures of Loranthaceae, as well as in suspension cultures of other plant
species [50–52]. According to Fowler [53], sucrose administered to the culture medium
is not only oxidized but also converted into starch as storage. However, it has been seen
that, if further limitations of carbon source occur, this starch will not necessarily be used
by cell cultures. Regarding growth kinetics, the best biomass yield was achieved when
cultures were started from high-density inoculum. That result correlates with other reports.
According to Torres [54], the use of low inoculum densities leads to a lengthening of the lag
and exponential phases during growth. For each clone/culture medium there is a critical
initial inoculum density, below which the culture will not grow. Mustafa [55] defined a
general protocol for the establishment of cell suspensions, in which the following inoculum
densities were determined: low (40–60 g PF L−1 ), medium (100–160 g PF L−1 ), and high
(>200 g PF L−1 ). Álvarez [56] reached the highest biomass values in suspensions of S. elaeagnifolium starting from 20% (v/v) inoculum density (GI = 4) in MS medium with 50 µM
NAA and 0.25 µM KIN. In the case of Tilia americana, batch suspensions (GI = 4.81 ± 0.88,
dt = 6.603 ± 0.78 d and µ = 0.107 ± 0.011 d−1 ) were started from 6% (v/v) inoculum
density in MS medium supplemented with 2 mg L−1 2,4-D and 0.5 mg L−1 KIN [57]. In
Capsicum baccatum, the maximum GI (3.11) was achieved when using modified MS with the
addition of 2,4-D (1.14 mg L−1 ) and BAP (0.23 mg L−1 ), with 12.5 g L−1 inoculum size [58].
Biomass doubling time varies with the species and culture conditions; for example, it was
60 h for Acer pseudoplatanus, 48 h for N. tabacum, 36 h for Rosa sp., and 24 h for Phaseolus
vulgaris, among others [59]. It is noticeable that L. cuneifolia suspension cultures, under
the conditions tested here, showed limited growth when compared with those reported
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for other plant species. It is necessary to continue optimizing other variables, such as the
carbon source, the base culture medium, or the addition of other combinations of growth
regulators to achieve better biomass yields for its subsequent scaling up to the bioreactor.
Regarding antioxidant activity, we found higher values in wild plant extracts, where
polyphenol contents were also higher when compared to in vitro cultures. These in vitro
results correlate with the in vivo and ex vivo experimental designs previously performed
in L. cuneifolia [11]. In accordance with our findings, a linear correlation between the total
phenolic level and antioxidant properties was described in V. album, which was attributed
to the presence of phenolic compounds in earlier studies [28,60].
The Ames test is used to evaluate the mutagenic activity of a given chemical. In
addition, it can be adapted for the detection of “protectants” or substances that decrease
mutagenic action. We did not detect mutagenic or anti-mutagenic activity in any case.
4. Materials and Methods
4.1. Dynamics of Polyphenols in Different Organs and Phenological Stages of Wild Plants
4.1.1. Plant Material
Plant material of L. cuneifolia (2–4 years old) growing on Vachellia caven (Mol.) Mol.
was collected from Villa de Merlo, San Luis (32◦ 21′ 22.5′′ S, 65◦ 00′ 20.5′′ W). A specimen was
deposited, under the name BAF 9018, in the herbarium of the “Juan Aníbal Domínguez”
Pharmacobotany Museum at the Faculty of Pharmacy and Biochemistry, University of
Buenos Aires. In 2018, branches of healthy specimens of L. cuneifolia were cut during the
flowering, fruiting, and post-fruiting seasons (four, six, and five specimens, respectively).
Flowering corresponded to the months of March and April (mean temperature 19.3–14.8 ◦ C,
air humidity 75.8–81.3%, and rainfall 11.8–3.6 mm), fruiting corresponded to the months of
November (mean temperature 20.5 ◦ C, air humidity 65.9%, and rainfall 17.19 mm), and
post-fruiting to December (mean temperature 22.1 ◦ C, air humidity 63.8%, and rainfall
69.8 mm). Finally, they were dried at room temperature in a dark and aerated area
for approximately one week and were stored in a desiccator until they were used for
polyphenol extraction. For histochemical analysis, fresh material was used. For that,
medium- to large-size branches of L. cuneifolia-containing fruits were cut and moistened
paper towels were placed around the cutting site. These were placed in sealable plastic
bags and transferred to Ciudad de Buenos Aires in camping refrigerators or polystyrene
boxes containing cooling gels, to keep the material as fresh as possible until its arrival at
the laboratory, where it was kept at 4 ◦ C until processing.
4.1.2. Polyphenol Extraction
Dry leaves, stems, and flowers from adult wild specimens were separately ground in
a rotary knife grinder (IKA). In both cases, a three-stage extraction was performed, first
with pure methanol, then with 80% (v/v) methanol, and finally with 50% (v/v) methanol.
Each stage was performed at room temperature for 24 h. The supernatants were pooled
and stored at −20 ◦ C for further analysis.
4.1.3. Spectrophotometric Analysis
•
Total phenolics content
Total phenolic content was determined using a modified Makkar [61] procedure.
Briefly, 100 µL of the sample (calli extracts or plant extracts) was mixed with 100 µL of
Folin–Ciocalteu reagent and kept in the dark at room temperature for 3 min. Then, 100 µL
of 0.3 N sodium carbonate was added to the mixture. The mixture was then further
incubated in the dark at room temperature for 30 min. The procedure was performed in
96-well microplates (Nunc™). The absorbance of the complex was measured at 765 nm
using a microplate reader (µQuant, Biotek, VT, USA) and then compared to a standard
curve prepared with various concentrations of gallic acid (100 µg mL−1 stock solution). The
results were expressed in mg GA eq·g−1 DW. All experiments were performed in triplicate.
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•
Total flavonoid content
Total flavonoid content was determined using a modified Rafi [62] protocol. For calli,
50 µL of extract, 20 µL methanol, 10 µL of 10% (w/v) aluminum trichloride (AlCl3 ), 10 µL
of 1 M potassium acetate, and 120 µL of distilled water were added to each well of a 96-well
plate. For plant extracts, 25 µL of extract, 45 µL methanol, 10 µL of 10% (w/v) AlCl3 , 10 µL
of 1 M potassium acetate, and 120 µL of distilled water were added to each well of a 96-well
plate. Then, the mixture was homogenized and incubated for about 30 min in the dark. The
absorbance of the solution was measured at a wavelength of 415 nm in a microplate reader
(µQuant, Biotek, VT, USA). Total flavonoid content was calculated with a QE calibration
curve (stock solution 1 mg QE mL−1 in methanol) and the results were expressed as mg
QE eq·g−1 DW. All experiments were performed in triplicate.
•
Hydroxycinnamic acid content
HCA content was determined using a modified Ricco [63] procedure. Briefly, 15 µL of
samples and 285 µL of ethanol were added to each well of a 96-well plate. Absorbance was
measured at 328 nm in a microplate reader (µQuant, Biotek, VT, USA) and then compared
to a calibration curve prepared with various concentrations of chlorogenic acid (1 mg mL−1
in absolute ethanol stock solution). The results were expressed as mg chlorogenic acid
eq·g−1 DW. All experiments were performed in triplicate.
•
Proanthocyanidin content
PA content was determined by a modified Horszwald and Andlauer [64] method.
Briefly, 25 µL of samples mixed with 150 µL 4% (w/v) vanillin and 75 µL 32% (v/v)
hydrochloric acid were added to each well of a 96-well plate. Then, the mixture was
homogenized and incubated for about 15 min in the dark. Absorbance was measured at
500 nm in a microplate reader (µQuant, Biotek, VT, USA). Total PA content was calculated
with a calibration curve prepared with various concentrations of (+)-catechin. Results were
expressed as mg catechin eq·g−1 DW.
Statistical analysis: ANOVA and when corresponding a post hoc Tukey’s analysis
were performed. Jamovi [65] and R [66] statistical software were used; p < 0.05 were
considered significant.
4.1.4. Thin-Layer Chromatography (TLC) Analysis
The qualitative analysis of polyphenols by thin-layer chromatography (TLC) was performed using Silica Gel 60 (Merck) as stationary phase and ethyl acetate:formic acid:acetic
acid:water (100:11:11:23) as mobile phase. Flavonoids and HCA derivatives were revealed
with a 1% (v/v) methanolic solution of the NP reagent and PA with a 5% (w/v) vanillin:HCl
ethanolic solution [67].
4.1.5. HPLC-UV Analysis
Considering the results obtained by spectrophotometric methods, extracts from calli,
cell suspensions, and adult specimens were selected for HPLC-UV analysis [11]. Each
analyte was compared among organs of the same phenological stage or PGR. Mean values
followed by an asterisk and different letters represent statistically significant differences
(p < 0.05, Tukey’s multiple comparison test).
4.1.6. Histochemical Analysis
Cross sections of leaves, primary stems, and embryos were made with a sliding microtome. The sections were stained with 1% AEDBE methanolic solution for the detection of
flavonoids and HCA derivatives, or with 5% vanillin:hydrochloric acid for the detection of
PA [67]. All sections were observed under bright-field and fluorescence light microscopes.
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4.2. In Vitro Culture Initiation, Growth Kinetics, and Polyphenolic Content under Different
Culture Conditions
4.2.1. Light Influence on Calli Induction, Growth, and Polyphenolic Content
Plant material for in vitro cultures was collected, preserved, and shipped to the lab as
described in Section 4.1.1 (histochemical analysis). Fresh and mature fruits were disinfected
as previously described [19]. Then, approximately 70 embryos were transferred to glass
tubes (one per tube) containing control White modified medium (CWM = White medium
with 500 mg L−1 casein hydrolysate, 100 mg L−1 myo-inositol, B5 vitamins, and 4%
(w/v) sucrose) supplemented with 2.50 µM NAA, 9.20 µM KIN, and 6.5 g L−1 of agar
(micropropagation grade); pH was adjusted to 5.6–5.8. One half of the glass tubes were
cultured in a chamber with a 16/8 h (light/darkness) photoperiod given by fluorescent
lamps (Narva T8 LT 18 W/760-010 daylight, Germany; irradiance 13.5 µmol m−2 s−1 ) and
the other half in a chamber in darkness. Cultures were maintained at 24 ± 2 ◦ C. After
60 days, the frequency of calli production (%) was estimated. Then, the established calli
were transferred to identical fresh media and samples were taken once a week for 16 weeks
to determine fresh weight (FW) and dry weight (DW) and for polyphenol qualitative
and quantitative analysis as described in Sections 4.1.2–4.1.5. Calli were oven dried at
40 ◦ C until constant weight. Then, extraction was performed as described for wild plant
specimens (Section 4.1.2). Experiments were made in duplicate and statistically analyzed
by chi-square and Fisher’s tests with Jamovi software [65].
4.2.2. Influence of PGRs and Inoculum Size on Growth Kinetics and Polyphenolic Content
Calli were transferred to glass tubes containing CWM with the addition of 1-naphthalene
acetic acid (NAA), 3-indole acetic acid (IAA), or 2,4-dichloro phenoxy acetic acid (2,4-D) at
2.5, 5.0 or 10.0 µM. After four weeks of adaptation to the new media, the growth curve was
initiated in the identical fresh medium using “low” inocula (100 to 250 mg FW) or “high”
inocula sizes (250 to 500 mg FW; n = 29 for each treatment). Cultures were maintained in a
growing chamber at 24 ± 2 ◦ C, under a 16 h light photoperiod using fluorescent daylight
lamps as previously detailed. Samples were taken every seven days during 8 weeks to
measure FW and DW and for polyphenol qualitative and quantitative analysis as described
in Sections 4.1.3–4.1.5. Calli were oven dried at 40 ◦ C until constant weight. Then, extraction
was performed as described for wild plant specimens (Section 4.1.2). Growth index (GI),
specific growth rate (µ), and doubling time (dt ) were calculated according to Equations
(1)–(3), respectively. Statistical analysis was performed by ANOVA once the assumptions
were confirmed. In case of significance in ANOVA, a Tuckey’s post hoc test was carried
out. If lack of homoscedasticity was detected, a Welch’s heteroscedastic F-test [68,69] was
performed, with a Games–Howell post hoc test when necessary [70,71]. Jamovi and R
Studio software were used [65,66].
Equation (1): formula for calculating the growth index (GI). x: biomass at time t; x0 :
initial biomass
x
GI =
(1)
x0
Equation (2): formula for calculating the specific growth rate (µ). x: biomass at time t;
x0 : initial biomass; t: final time; t0 : initial time
µ=
ln ( xx0 )
t − t0
(2)
Equation (3): formula for calculating the doubling time (dt ). µ: specific growth rate
dt =
ln2
µ
(3)
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4.2.3. Establishment of Cell Suspension Cultures
Calli growing in semi-solid CWM with 2.50 µM NAA and 9.20 µM KIN as PGRs were
transferred to 150 mL Erlenmeyer flasks containing 30 mL of the same culture medium but
without agar. Two inoculum sizes were tested, 100 mg/flask (low) and 750 mg/flask (high).
The flasks were transferred to an orbital shaker at 110 rpm, 24 ± 2 ◦ C, and maintained
in the dark. Samples were taken on days 7, 14, 21, and 28 to determine viability (Evans
blue and fluorescein diacetate tests [72]), FW, and DW and for polyphenol qualitative and
quantitative analysis as described in Sections 4.1.3–4.1.5. Cell suspensions were filtered
and dried at 40 ◦ C until constant weight. Then, extraction was performed as described for
wild plant specimens (Section 4.1.2).
4.3. Antioxidant Activity of Wild Plants and In Vitro Cultures Extracts
Wild plant and in vitro culture extracts were obtained as described in Section 4.1.2.
Antioxidant activity was determined according to the method of Cheng [73] by using
2,2-diphenyl-1-picrylhydrazyl (DPPH; a radical organic compound with two absorption
peaks: 340 and 515 nm) that reduces its absorption at 515 nm in the presence of antioxidant
compounds. Results were expressed as mg trolox eq·g−1 DW and descriptive statistical
analysis was performed
4.4. Mutagenicity and Antimutagenicity Assays of Wild Plants and In Vitro Culture Extracts
4.4.1. Mutagenicity Assay
The Ames test was carried out to evaluate the possible mutagenicity of the extracts of L.
cuneifolia wild plants and in vitro cultures. Salmonella typhimurium TA98 and TA100 strains
were used in order to detect frameshift mutations and base-pair exchanges, respectively [74].
Tests were carried out to confirm the phenotypic characteristics for resistance to ampicillin,
the rfa mutation (permeability to crystal violet), and the uvrB mutation (sensitivity under
UV light). The test was also carried out with and without the addition of S9 mix (metabolic
activator) by means of the plate incorporation method. The test is considered positive
when, at least, the number of spontaneous revertants is doubled. In tubes containing 2 mL
of soft agar (at 45 ◦ C) supplemented with L-histidine HCl-D-biotin (0.5 mM–0.5 mM), 50 µL
of a culture incubated for 12 h with a density of 1 × 108 cells mL−1 of the selected control
strains (TA98 or TA100), and 0.5 mL of S9 mix for the assay with metabolic activation or
phosphate buffer (pH 7.4) in the assays without metabolic activation. Then, 50 µL of the
dilution of the wild plant or calli extract was added. This mixture was gently homogenized
with a vortex and plated with a minimal medium for its incubation at 37 ◦ C for 48 h. Then,
the revertant His+ colonies were counted. In all the tests, negative controls were included
to know the rate of appearance of spontaneous revertant colonies. As positive controls, B1
aflatoxin (2 µg/plate) and 2-aminoanthracene (5 µg/plate) were used for the assays with
metabolic activation in both strains, and sodium azide (5 µg/plate) and 2-aminofluorene
(10 µg/plate) for assays without metabolic activation, in TA 100 and TA 98, respectively.
Results were expressed as the reversion coefficient or C.R. = No. of revertant colonies per
assayed plate/No. of revertant colonies per control-spontaneous-plate.
4.4.2. Antimutagenicity Assay
A volume of 0.5 mL of S9 mix, 50 µL of B1 aflatoxin, and 50 µL of tester strain
suspension was placed in each tube and brought to a final volume of 2 mL with water.
50 µL of water were added to the positive control, and 50 µL of wild plant extract to the
unknown sample. A blank without aflatoxin was also performed in which 100 µL of water
and an extract blank without B1 aflatoxin and with 50 µL of water were used. The results
were statistically analyzed using the Bartlett test to determine the homogeneity of variances
and then compared using the Kruskal–Wallis test.
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4.5. Chemicals and Reagents
White medium [75], sucrose, casein hydrolysate and agar (plant tissue micropropagation culture grade) were from PhytoTechnology Laboratories (Lenexa, KS, USA). LiChrosolv®
Methanol was supplied by Merck (Darmstadt, Germany). Formic acid was purchased from
Baker (New Jersey, USA). Ultrapure water was generated with a Barnstead Thermo Scientific™ (Waltham, MA, USA). Quercetin-3-rhamnoside (Q-3-O-Rh) was from Extrasynthese
(Lyon, France); catechin (C), quercetin-3-O-glucoside (Q-3-O-G), quercetin-3-O-xyloside
(Q-3-O-X), quercetin-3-O-arabinofuranoside (Q-3-O-AF), quercetin-3-O-arabinopyranoside
(Q-3-OAP), and chlorogenic acid (CA) were from Sigma (St. Louis, MO, USA). The other
chemicals, standards, and solvents were purchased from Sigma-Aldrich (Saint Louis,
MO, USA).
5. Conclusions
Regarding wild plants, the post-fruiting stage proved to be the best time of harvesting
to obtain the maximum amounts of polyphenolic compounds. Further experiments will be
performed to continue analyzing the influence of different host plants on the polyphenolic
profile. L. cuneifolia appeared to be a very challenging species to be introduced in in vitro
culture. Different PGR ratios, light conditions, and inoculum sizes were tested, and only
a slight improvement in growth and polyphenolic content was achieved. We established
that, among the conditions tested, calli grew and produced FL, HCA, and PA in CWM
with 2.5 µM IAA and 16 h photoperiod, but at low levels compared to wild plants. As for
cell suspension cultures, the conditions tested were not successful either for growth or for
polyphenolic production. Other media components such as carbon and nitrogen source
and PGR ratios must be assayed to establish the optimal conditions to obtain cultures with
better growth. Then, other strategies such as elicitation or two-step cultures (culturing cell
suspensions in a medium that promotes growth and then transferring them to a medium
that favors polyphenolic production) could be examined to obtain improved yields. No
mutagenic or antimutagenic activity was detected in wild plants or in in vitro culture
methanolic extracts.
Author Contributions: Conceptualization, M.V.R., M.L.W., R.A.R. and M.A.Á.; methodology, M.V.R.,
M.L.B. (Martín León Bari), M.L.B. (Melina Laguia Becher), A.V.C., P.L., C.B.D. and S.A.T.; formal
analysis, M.V.R., M.L.W., C.B.D., R.A.R. and M.A.Á.; investigation, M.V.R., M.L.W., R.A.R. and
M.A.Á.; resources, A.P.; data curation, M.V.R., A.V.C., P.L., C.B.D. and S.A.T.; writing—original draft
preparation, M.V.R. and M.A.Á.; writing—review and editing, M.V.R., M.L.W., C.B.D. and M.A.Á.;
supervision, M.L.W. and M.A.Á.; project administration, M.A.Á.; funding acquisition, M.L.W. and
M.A.Á. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the ANPCyT (grant number PICT 2015–2024), UBACyT (grant
number 20020170100121BA), and Universidad Maimónides. M.A.A. and M.L.B. (Melina Laguia
Becher) are members of CONICET, MVR and M.L.B. (Martín León Bari) have a Ph.D.-co-financed
grant from CONICET—Universidad Maimónides.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Acknowledgments: We wish to thank the biochemist Guadalupe M. Vedoya for their contribution in
mutagenicity tests and Leonardo Ulises Spairani for their contribution in statistical analysis.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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