1. Introduction
Medicinal plants, also referred to as herbal plants, have been used for centuries as a source for alternative medicines in the prevention and treatments of various diseases. Globally, plant-based products have been well-accepted due to scientific evidence that proved their low to no side effects and low price compared to modern medicine [
1]. Presently, approximately 70–80% of the world’s population depend on plant-based products as their primary healthcare needs [
2]. In Malaysia, the herbal industry has become one of the most important sectors with RM 80 million (USD 18 million) production values in 2018. With an ever increasing demand, the value has been projected to increase manifolds [
3].
Clinacanthus nutans, commonly known as Sabah snake grass, is a medicinal plant species belonging to the family Acanthaceae and is among the more important medicinal plants in Malaysia. It is an annual shrub which grows up to three meters tall with lanceolate-ovate shaped leaves [
4]. The species is being widely used in Southeast Asian countries like Malaysia, Thailand and Indonesia as a remedy for the treatment of herpes simplex virus, varicella-zoster virus, fever, diabetes and as anti-venom for snake and scorpion bites [
5,
6]. Dried leaves of
C. nutans are also known to treat hepatitis infection, fever, eczema, shingles and inflammation [
6,
7,
8,
9]. The medicinal properties of
C. nutans leaf extracts have been recognized for many generations and have been commonly used in traditional medicines. According to the Ministry of Agriculture and Agro-based Industry Malaysia,
C. nutans has been selected under the National Key Economic Area (NKEA) research grant schemes alongside 17 other herbal plants. They were selected based on their potentials for commercialization [
10]. Meanwhile, in Thailand, the National Drug Committee recognized
C. nutans as one of Thailand’s National List of Essential Medicines in 2011 [
11].
Presently, the extraction of bioactive compounds or plant secondary metabolites from plant-based sources has gained attention in pharmaceutical industries. Advances in plant biochemistry and herbal medicines have identified several bioactive compounds in
C. nutans leaf extracts, namely stigmasterol, kaempferol, quercetin, gallic acid, apigenin and vitexin. Additionally, several unique bioactive compounds have also been identified such as clinacoside (A, B, C), cycloclinacoside (A1, A2), clinamide (A, B, C, D, E), 2-cis-entadamide A and entadamide (A, C) [
8,
12,
13].
Generally, natural products are the most preferred source of antioxidants in disease prevention than synthetic antioxidants due to them having few side effects. The demand for the raw material of
C. nutans has led to the over-harvesting of the wild species which has significantly reduced their availability in nature [
4]. Hence, a biotechnological approach in the propagation of the plant through an in vitro technique is perceived as an alternative method for the large-scale production of the raw materials in a shorter time. Plant growth regulators, the basal medium strength and sucrose concentration largely influence the success of an in vitro technique for plant propagation. Through this technique, the phenolic contents and antioxidant activities are also known to be affected. The present study was undertaken to investigate the effects of plant growth regulators, basal medium strength and sucrose concentrations of in vitro culture of
C. nutans with the aim of optimizing the basal medium for the mass propagation of plants. The study also was conducted to analyze the phenolic contents and antioxidant activities between the leaves of tissue-cultured and conventionally propagated
C. nutans as affected by different aqueous extraction temperatures.
3. Discussion
In this present study, the optimum basal medium formulation by the alteration of plant growth regulators, basal medium strength and sucrose concentration for the in vitro propagation of
C. nutans were established. In plant tissue culture, plant growth regulators are one of the most important factors affecting the growth of explants. In a single node culture of
C. nutans, the shoot induction was affected by the application of cytokinin in the culture medium. The synthesis of cytokinin in plants becomes limited due to injury when plant parts are excised into smaller sizes [
15,
16]. Hence, cytokinin was supplemented in the basal medium to support shoot induction, cell division and cell differentiation. In the present study, 12 µM BAP was more favorable than other cytokinins in the initial stage of the shoot induction of
C. nutans. A study on
Justicia gendarussa (Acanthaceae) showed that the supplementation of BAP in an MS basal medium exhibited the highest percentage of shoot induction, number of shoots and shoot length, compared to a basal medium supplemented with kinetin and thidiazuron (TDZ) [
17,
18]. In addition, similar studies on
Andrographis paniculate, which belong to the same family as
C. nutans and
J. gendarussa, also recorded that the application of BAP was able to produce a higher percentage of shoot regeneration and number of shoots [
19].
The integration of cytokinin and auxin is the most commonly used technique in plant tissue cultures to increase the multiplication and proliferation rates of plantlets. However, the present study observed that the application of auxin significantly reduced the percentage of shoot regeneration, number of shoots, length of shoots, number of leaves and fresh weight of leaves. A supplementation with auxin resulted in the production of a massive amount of callus. Auxin is supplemented in plant tissue culture basal medium to facilitate cell elongation and induce rooting. In several cases, due to the presence of endogenous auxin in plants, callus is formed [
20]. The current findings on the effects of auxin were also supported by a previous study on
Tylophora indica in which a combination of BAP and IBA in as low as 1.23 µM of a basal medium significantly reduced the growth of the plant compared to same cultured in BAP alone [
21]. Similarly, another study on
Spilanthes acmella Murr. reported that the supplementation of cytokinin alone in the basal medium produced better growth [
22]. In both of these studies, the callus formation was observed in the basal regions of the explants [
21,
22].
The effect of the basal medium strength on the in vitro growth of
C. nutans was also investigated. The full-strength MS basal medium yielded superior results for all the parameters studied. Similar results were also reported in a previous study by Chen et al. [
23], where a full-strength MS basal medium supplemented with 4.44 µM BAP and 0.11 µM NAA produced the highest multiplication rate and the longest shoots for
C. nutans. The study reported that as the basal medium strength was reduced to half, the number of leaves and length of shoots significantly decreased. Besides, the growth rate slowed and the explants stunted. Based on the growth vigor and morphological changes, the full-strength basal medium produced more vigorous shoots and greener leaves compared to half-strength basal medium which produced yellowish leaves. The full-strength basal medium was also found to be most effective for the micropropagation of
Ocimum basilicum [
24],
Bacopa monnieri [
25] and
Thymus hyemalis [
26]. All of these findings were in agreement with current findings, indicating the effectiveness of the full-strength MS basal medium for the medicinal plant studied. On the contrary, the double-strength MS basal medium appeared to be a toxic basal medium, while quarter- and half-strength MS basal media were found to cause nutrient deficiencies in explants [
24]. The MS basal medium is known to be a balanced culture medium in terms of macronutrients, micronutrients and vitamins suitable for a wide range of plant species.
A continuous supply of carbon is important in plant tissue cultures in order to support plant growth. Several studies have been conducted on different species of plants. In a study by Naz et al. [
27], carbon sources including sucrose, glucose and fructose at concentrations from 10 to 50 g/L were supplemented in the basal medium of
Althaea officinalis. The results indicated that 30 g/L of sucrose exhibited the best result for all the parameters measured. Similarly, Reddy et al. [
28] reported similar results for
Ceropegia ensifolia. The addition of 30 g/L sucrose was found to be the optimum for the growth of
Thymus hyemalis compared to other types of carbon sources including glucose, fructose, mannitol and sorbitol [
17]. The studies implied that a sucrose concentration higher than 30 g/L results in a decrease in the multiplication and growth rate of plants [
24,
25,
26,
27,
28,
29,
30,
31,
32,
33,
34,
35,
36,
37,
38]. These findings were in agreement with the present study in which 30 g/L of sucrose was established as the optimum for the growth of
C. nutans. The literature has previously found that sucrose is found to be the most suitable carbon source for in vitro propagation compared to glucose, fructose and other types of sugar, and is the most common carbohydrate present in the phloem saps of plants. Hence, the translocation process in the plant will be eased [
29,
30]. The addition of sucrose at more than the optimum level would increase the osmotic pressure of the basal medium, causing the water and mineral uptake by explants to be inhibited. Gas exchange in culture vessels would be limited resulting in an increased toxicity of the basal medium [
31].
In the present study, the propagation technique (tissue culture and conventional propagation) and the effects of aqueous temperatures (25 and 100 °C) on the extraction of phenolics contents and antioxidant activities were studied. Based on the total polyphenols, total phenolic acids and total flavonoids contents measured, tissue-cultured leaves significantly produced higher values on all the parameters under study compared to the conventionally propagated leaves. The extractions at 100 °C recorded a higher amount of total polyphenols, total phenolic acids and total flavonoids from both sources of leaves compared to extractions at 25 °C. Pandey et al. [
32] conducted a study on
Origanum vulgare, comparing the leaves of the tissue-cultured plant and conventionally propagated mother plants. They found that the leaves of tissue-cultured plants significantly produced higher total phenolics, flavonoids and tannins contents than the leaves of the mother plant. In another study comparing the field-grown and tissue-cultured plants of
Lavandula angustifolia var., Ellagance Blue, Blue River and Munstead found that tissue-cultured plants of all three varieties exhibited a higher total polyphenols content compared to field-grown plants [
33]. A study by Bose et al. [
34] reported a similar finding on mother and tissue-cultured plants of
Nardostachys jatamansi extracted with different extraction solvents including methanol, acetone, chloroform, acetonitrile and water. Their results showed that the leaves of tissue-cultured plants significantly produced higher total phenolics, flavonoids, alkaloids and tannins contents in all the extraction solvents tested. The variations in phenolics content could be due to different extraction processes such as different extraction temperatures and different sources of plants. The concentration of secondary metabolites in plants could be affected by hormonal contents as well as endogenous physiological changes in the plants. According to Umebese and Falana [
35], the accumulation of the phenolics content is generally low when plants are grown under nonstress conditions. The accumulation of phenolics increases when plants are exposed to stress conditions.
In an antioxidant activities study, it was observed that leaves from tissue-cultured plants exhibited higher DPPH, ABTS and FRAP values compared to leaves from conventionally propagated plants. Studies by Amoo et al. [
36] and Bhattacharyya et al. [
37] on
Aloe arborescene and
Dendrobium thyrsiflorum, reported similar findings. The strong antioxidant activities in plant extracts could be due to the presence of high concentrations of secondary metabolites such as phenolics, tannins and alkaloids contents. High concentrations of secondary metabolites in plant samples enhance their redox properties and the ability to scavenge free radicals [
38,
39].
A study on superoxide anion radical scavenging activities showed that leaves from tissue-cultured plants produced a higher inhibition percentage in comparison with leaves from conventionally propagated plants. Similar findings were reported on shoot and root extracts of tissue-cultured
Salvia miltiorrhiza, producing a higher inhibition percentage of superoxide anion radical scavenging activity compared to extracts from seed-derived plants [
40]. Superoxide radicals are known to be extremely harmful as they can act as a precursor for the production of reactive oxygen species. An excessive amount of superoxide anions produced in cells increases dismutation and leads to hydrogen peroxide production and thereby increases the oxidative stress levels in the human body [
41]. Hence, the ability of plant samples to scavenge superoxide anion is important to reduce the concentration of superoxide radicals and lower oxidative stress levels.
The results in the present study on the phenolics content and antioxidant activities recorded that aqueous temperature of 100 °C was more prominent in producing a higher phenolic content and antioxidant activities compared to the aqueous temperature of 25 °C. The results showed that the coefficient of diffusion and phenolic contents solubility increased as the aqueous temperature was increased. An increased solubility of phenolic contents by a higher temperature favors the release of bound phenolics in samples with cellular constituents of the plant cell breakdown which leads to an increase in the cell membrane permeability [
41]. In addition, an elevated extraction temperature causes the rate of thermally stable antioxidants to be higher than the rate of decomposition of less soluble antioxidants [
42]. This was proven by 100 °C aqueous extraction of
C. nutans leaves to gain higher antioxidant activities.