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,
10,
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,
22,
23]. Accordingly, in this study, we tested different PGRs on calli growth and polyphenol yield over time. In 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.
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, 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,
30,
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 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,
48,
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,
51,
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, d
t = 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 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.