1. Introduction
For centuries, indigenous people have recognized the role of fire and smoke on plant cultivation and productivity [
1,
2]. In an attempt to explore this knowledge under standard conditions, scientists have simulated this phenomenon by generating smoke in a drum using compressed air and bubbling through distilled water to form smoke-water (SW). Smoke and fire (smoke-technology) holds great potential in various agricultural and biological fields, and the scientific evidence on their positive role has been demonstrated in several plants [
3]. The active compound was successfully isolated and identified as karrikinolide (KAR
1), previously referred to as butenolide; this has resulted in its increased interest by researchers globally [
1,
2,
3,
4,
5,
6]. Given that no two batches of SW contain exactly the same balance or concentration of compounds, the isolation of active compounds in SW eliminates the disparity and ambiguity often associated with SW in crude solution [
7]. Presently, several types of KAR, generally referred to as karrikins, have been identified, and are recognized as a new family of plant growth regulators (PGRs) [
8]. Both SW and KAR
1 are known to interact with other PGRs [
8] and often exhibit cytokinin and auxin-like activities, as demonstrated in the mungbean bioassay [
9]. The use of different PGRs, especially cytokinins, has been demonstrated to be a vital elicitor of valuable phytochemicals in medicinal plants [
10,
11,
12]. Thus, SW and karrikins hold great potential as useful tools for enhancing plant productivity, given their influence on plant growth and development, as well as on biochemical pathways, including the phenylpropanoid pathway that serves as a rich source of metabolites in plants [
2,
13,
14,
15]. In recent times, phytochemicals have received increasing attention due to their antioxidant properties and ability to counteract oxidative stress associated with various diseases [
16]. As a result, these bioactive phytochemicals have been widely-explored for their therapeutic and pharmacological (e.g., antioxidant, antimicrobial, anti-inflammatory, and anti-diabetics) value, as evidenced in several African medicinal plants [
17].
In folk medicine, the genus
Eucomis, including
Eucomis autumnalis subspecies
autumnalis (family: Asparagaceae), is a popular remedy against a variety of diseases [
18]. For instance, it is administered as an enema to treat lower backache, biliousness, and urinary diseases, as well as fevers and fractures, among the Zulus in South Africa [
19]. Recently, there have been increasing concerns about the conservation status of members in the genus
Eucomis due to their endemic nature, indiscriminate harvesting, and wide utilization, particularly the underground parts such as the bulbs and roots [
18]. The potential of using micropropagation as a means to ensure the sustainability of members of the genus has been recognized [
18,
20]. However, the quality and quantity of the bioactive phytochemicals in micro-propagated clones needs to be guaranteed to gain acceptance by different stakeholders, such as the consumers and traders in local communities, as well as herbal-based industries (nutraceutical and pharmaceutical companies) which are interested in these plants [
21,
22].
In most cases, extensively sought-after plants are often collected from the wild, resulting in a decline in their natural populations [
17]. Researchers have recognized the need for effective conservation techniques for medicinal plants and devising approaches for the resupply of pharmacologically-active phytochemicals to meet the envisaged demands of the pharmaceutical industry [
20,
21,
22]. Micropropagation generally allows for the mass production of clonal plant materials in a relatively short time and the utilization of elicitors to facilitate the accumulation of different phytochemicals [
22]. In addition, an in vitro approach is often utilized to increase the biosynthesis and accumulation of antioxidant compounds in micropropagated plants [
23]. Given that the value of cultivated medicinal plants is often dependent upon the quantity and quality of the accumulated phytochemicals which determine their bioactivity [
21], research endeavors that can establish the integrity of micropropagated plants are desired. Thus, the current study evaluated the phytochemical content and antioxidant activity in in vitro regenerants and acclimatized
Eucomis autumnalis subspecies
autumnalis following treatment with SW and KAR
1. The current study was guided by the following research questions:
(1) How does the application of SW and KAR1 influence the phytochemical pool and antioxidant activity of Eucomis autumnalis subspecies autumnalis?
(2) What are the dynamics of the aforementioned parameters in in vitro and acclimatized plants?
(3) What are the differences in terms of the phytochemical and antioxidant activities of the aboveground and underground parts of acclimatized plants?
4. Discussion
Secondary metabolites in natural resources, including medicinal plants, have been widely explored for their biological properties [
22,
30], and members of the genus
Eucomis are well-known for their rich phytochemicals [
31]. Globally, there is concern for the increasing decline in valuable plants that are frequently collected from the wild, thereby causing severe strain and a decline of their natural populations [
17,
18,
20]. The need for sustainability of plants as sources of valuable, bioactive compounds cannot be overemphasized [
17,
22]. Even though there is still limited knowledge on the plants biosynthetic pathway and underlying mechanisms of the action involved in the production of the desired phytochemicals [
32], the use of elicitor(s) often influences their resultant integrity in terms of quality and quantity [
33]. The potential role of SW and KAR
1 on the phytochemical pool have been demonstrated in different plants, including
Musa species [
13],
Tulbaghia species [
34],
Isatis indigotica [
35], and
Aloe arborescens [
36]. In the current study, the inclusion of SW or KAR
1 in the growth media during the micropropagation stage had a significant effect on the resultant phytochemicals in in vitro and acclimatized
Eucomis autumnalis subspecies
autumnalis. The therapeutic effects of the majority of the phytochemicals quantified in
Eucomis autumnalis subspecies
autumnalis are well established [
22,
23,
37]. For example, ferulic acid is known to exhibit biological activities such as antioxidant and anti-inflammatory [
38], and this particular compound was one of the major phenolic acids in the roots and bulbs of the acclimatized plants. The increased concentration of ferulic acid observed in the roots and bulbs of the acclimatized plants, obtained from SW (1:1000 and 1:1500, dilutions) and KAR
1 (10
−7, 10
−8 and 10
−9 M) treatments, is noteworthy, given that this plant is used for inflammation-related conditions.
The presence of an elicitor is known to activate genes related to defense-systems which often trigger the biosynthesis and accumulation of secondary metabolites [
33]. This is also supported by the fact that in vitro propagation systems create some degree of abiotic stress. However, the transfer of the micro-propagated regenerants to ex vitro conditions is known to cause changes in the quality and quantity of secondary metabolites in plants [
11,
39,
40]. This may be due to the utilization of some of the early-produced secondary metabolites as precursors for the biosynthesis and accumulation of other metabolites as the plant goes through different physiological stages over time [
12,
30,
39]. The presence of higher concentrations of phenolic acids in in vitro regenerants than in acclimatized plants have been demonstrated in
Merwilla plumbea [
40],
Eucomis autumnalis subspecies
autumnalis [
11], and
Artemisia judaica [
41]. Among the nine phenolic acids quantified in
Eucomis autumnalis subspecies
autumnalis, the concentrations of approximately 70% of the hyroxybenzoic (protocatechuic,
p-hydroxybenzoic and vallinic acids) and hydroxycinnamic (coumaric, ferulic and cinnamic acids) derivatives were several times higher in the in vitro regenerants than in the acclimatized plants. However, eucomic acid was generally higher in the acclimatized plants, while no discrete pattern was observed with the concentrations of flavonoids at different plant stages.
Eucomic acid has been quantified in medicinal plants such as
Eucomis autumnalis [
28,
42],
Opuntia ficus-
indica [
43], and
Cryptostephanus vansonii [
44]. Previously, Okada et al. [
45] isolated eucomic acid from
Lotus japonicus; this was considered a potential leaf-opening factor (
LOF) in this species. Likewise, the growth inhibitory potential of eucomic acid isolated from
Cattleya trianaei was demonstrated at varying concentrations [
46]. In the current study, eucomic acid was one of the phytochemicals that was abundant across the different treatments, regardless of the development stage. This observation suggests the wide distribution of eucomic acid in medicinal plants, and may be considered a marker compound in some species, especially for members of the genus
Eucomis. Although no significant increase in the levels of eucomic acid was observed in in vitro plantlets treated with SW and KAR
1, the leaves of the acclimatized plants, especially those from SW (1:500 and 1:1500) treatment, had significantly higher eucomic acid contents relative to the control. Despite the occurrence of eucomic acid in a large number of plants, especially in members of the genus
Eucomis, evidence of their specific biological activities remains speculative.
Several pharmacological activities, including antioxidant potency, are often attributed to the quality and quantity of phenolic acids and flavonoids in plants [
16,
30,
37]. Apart from being an important class of compound used for preventing many diseases, antioxidants play a crucial role as food additives to counteract spoilage caused by oxidizable nutrients [
23,
37]. The antioxidant activity of natural products is often evaluated via multiple methods that entail different mechanisms such as the hydrogen atom transfer (HAT) and single electron transfer (SET) [
47,
48]. As a result, the extracts of
Eucomis autumnalis subspecies
autumnalis were evaluated using DPPH and
β-carotene assays in order to establish the influence of SW and KAR
1 applications under diverse conditions. In the current study, the extracts generally demonstrated higher antioxidant potential in the
β-carotene model than in the DPPH assay. The highest antioxidant power (
β-carotene model) was observed in KAR
1-treated in vitro regenerants, i.e., almost two-fold higher than the control. Based on the responses in the in vitro and acclimatized plants, whereby majority of the extracts had higher antioxidant activity in
β-carotene model when compared to the DPPH assay, the mechanism of the antioxidant activity of
Eucomis autumnalis subspecies
autumnalis is more likely to be inclined towards HAT than SET. Similar findings were also exhibited by the extracts of micropropagated and acclimatized plants that were treated with different PGRs at varying concentrations [
11,
12,
49]. Plants are generally known to synthesize and accumulate secondary metabolites at varying concentrations in their different organs [
23], and this may influence the resultant antioxidant potential. However, it is often difficult to directly link the phytochemical pool to the antioxidant activity of medicinal plants [
11,
12,
40,
49].
In the current study, the variation observed in the antioxidant activity of different parts of acclimatized plants is important from a conservation perspective. For instance, the use of alternative plant organs, with a minimal detrimental effect on the survival of the whole plant, has been strongly recommended by researchers [
17]. This means that the higher antioxidant activity exhibited by the leaves of acclimatized
Eucomis autumnalis subspecies
autumnalis provides a valuable alternative to the underground plant parts which are widely utilized in traditional medicine. The current findings also suggest that the biological effects of medicinal plants often differ based on the plant part investigated [
10,
18,
44].
5. Conclusions
The importance of SW and KAR1 as potential elicitors for bioactive phytochemicals was demonstrated in Eucomis autumnalis subspecies autumnalis. This may provide an alternative approach for the production of secondary metabolites with therapeutic potential. Based on the use of UHPLC, the phytochemical profiles of Eucomis autumnalis subspecies autumnalis treated with SW and KAR1 was established for in vitro and acclimatized plants. Generally, in vitro regenerants accumulated higher concentrations of phytochemicals which significantly decreased after plants underwent prolonged periods of continuously-changing climatic conditions in the greenhouse. Among the nine phenolic acids in the in vitro regenerants, coumaric acid was the major (23-52 μg/g DW) bioactive compound. Acclimatized plants had only six types of phenolic acids, including syringic acid, which was absent in the in vitro stage. Likewise, the levels of a number of flavonoids were generally low, and different types were accumulated in in vitro and acclimatized plants. The levels of eucomic acid, which can be considered a diagnostic compound in Eucomis species, was significantly accumulated in the leaves of SW (1:1500) treatment after acclimatization. Antioxidant activity was relatively higher in the acclimatized plants when compared to the in vitro regenerants. Given the limitations associated with the two test systems used in the current study, the antioxidant activity demonstrated by the extract may be considered to be of low clinical significance. Thus, other test systems, especially in vivo systems, will be essential to reach a valid conclusion about the antioxidant potential of SW and KAR1-treated plants. From a conservation perspective, the current findings provide preliminary evidence of the value of SW and related technology as a potentially-viable method for the biosynthesis of phytochemicals of therapeutic importance in medicinal plants. Nevertheless, it will be necessary to establish the carry-over effect of SW and KAR1 for a longer duration (>1 year) on the phytochemical pools and other pharmacological activities (besides antioxidant), as well as to determine the overall safety of plant extracts. In addition, it will be necessary to investigate other medicinal plants in order to reach a valid conclusion about the overall potential of the tested compounds.