Influence of Multiwalled Carbon Nanotubes and Biostimulators on Growth and Content of Bioactive Constituents of Karkade (Hibiscus sabdariffa L.)

This work was carried out to evaluate the effect of different concentrations of multiwalled carbon nanotubes (MWCNT), 0, 500, and 1000 mg/L, and biostimulators (Delfan plus), 0 and 10 ml/L, and their interactions (sprayed on the plant shoot and soil until the complete wetness) on growth and production of bioactive constituents of karkade (Hibiscus sabdariffa L.) planted in plastic pots. After finishing experiment, growth parameters (length of shoot and root, number of leaves, branches, and fruits, chlorophyll content, and dry weight of calyces yield) were measured and also the active compounds of aqueous extracts calyces were measured by Gas Chromatography-Mass Spectrometry (GC-MS). The obtained results explained that the experimental treatment caused a significant increase in all measured parameters. GC-MS analysis shows quantitative and qualitative alterations of bioactive constituents of water extract from calyxes of karkade, where plant production of 60 active compounds at the combination of 1000 mg/L of MWCNT and 10 ml/L of Delfan plus was compared to untreated plants which produced 4 only.


Introduction
Medical plants researches have been expanded apparently during the last decade and international trend towards the use of natural plant remedies increased the need for more information about the properties and uses of medicinal plants. Hibiscus sabdariffa L., commonly known as roselle (English) and karkade (Arabic), belongs to Malvaceae and is an important annual crop grown successfully in subtropical and tropical climates [1]. The calyxes of karkade are rich in bioactive compounds including organic acids, phenolic compounds, vitamin A and vitamin C [2], flavonoid (anthocyanins), gossypetin, quercetin, pectin [3], saponins, glycosides, and alkaloids [4]. Additionally, Nnam and Onyeke [5] mentioned that calyxes contain 6.4% protein, 79.25% carbohydrates, 5.13% fat, 2.7% crude fiber, and 6.52% ash. Fresh or dried calyxes of karkade have been used traditionally as herbal drinks (cold and hot) because karkade has different therapeutic properties such as anticancer, antiviral, antifungal, antibacterial, antioxidant, antidiabetic, antihypertensive, antihyperlipidemia (anticholesterol), antiobesity, antispasmodic, and antitumor effects, decreasing the viscosity of the blood and stimulating intestinal peristalsis, and can be used to treat sore throats, coughs, and genital problems and it has also nephro/hepatoprotective and renal/diuretic effect and so forth [6].
In the recent years, there has been an increasing interest in nanoagriculture; the use of nanotechnology in agriculture and field application are highly important and are widely used in agricultural sciences, especially in the fields of biotechnology and tissue culture. The uptake of carbon nanomaterials by plants has shown a very recent field of nanoagriculture. Nanotubes are capable of interacting with biomolecules and creating functional nanosystems for transportation of other materials within cells, which leads to interaction of nanotubes and other compounds at morphological, cellular, and even molecular levels [7]. The application of biostimulators for different plants has positive effect on plant development due to containing high levels of free amino acids, total nitrogen, organic matter, and organic carbon [8].   Therefore, in the view of the medicinal importance of karkade calyxes, this work aimed to evaluate the effect of different concentrations of multiwalled carbon nanotubes and biostimulators (Delfan plus) on growth and quantitative and qualitative alterations of bioactive constituents measured by GC-MS.

Materials and Methods
A pot experiment was conducted in Biology Department, College of Science, University of Al-Qadisiyah, which included planting seeds of karkade plant in 18 plastic pots (5 seeds per pot) on 15/02/2016, and then the developing plants were reduced to one plant per pot. The experimental treatment was sprayed on the plant shoot and soil until the complete wetness; multiwalled carbon nanotubes (MWCNT) ( Table 1) were added at three concentrations, 0, 500, and 1000 mg/L, on 10/05/2016. Biostimulators (Delfan plus) ( Table 2) were added at two concentrations of 0 and 10 ml/L on 15/5/2016. The addition of two factors was repeated after one month, respectively.
After finishing the experiment (01/11/2016), growth parameters were measured, including length of shoot and root, number of leaves, branches, and fruits, dry weight of calyxes yield, chlorophyll content (measured by chlorophyll meter 502 SPAD), and the content of bioactive constituents per plant after preparation of aqueous extract of the calyxes of karkade harvested during periods of experience and then those bioactive constituents were measured by GC-MS technique (manufacturer: Shimadzu, GCMS-QP2010 Ultra system comprising a AOC-20i, Japan), according to the method of Srinivasan et al. [9].

Statistical Analysis.
Data analysis was carried out using Randomized Complete Blocks Design (RCBD) in a factorial arrangement (3 × 2) with three replications per each treatment. Revised Least Significant Difference (RLSD) was used to compare treatment means at probability level of 0.05 when treatment effect was significant [10].

Results
The statistical analysis of Table 3 elucidates that increasing concentrations of both multiwalled carbon nanotubes (MWCNT) and biostimulators (Delfan plus) added to the plants alone or with each other lead to a significant increase in all studied parameters of karkade (length of shoot and root, number of leaves, branches, and fruits, chlorophyll content, dry weight of calyxes yield, and number of bioactive constituents) in comparison with those obtained from untreated plants; using the combination of 1000 mg/L MWCNT and 10 ml/L Delfan plus caused the highest increase which reached 30.47% length of shoot, 45.73% length of root length, 64.89% number of leaves, 62.08% number of branches, 60.98% number of fruits, chlorophyll content of 13.77%, and 48.30% dry weight of calyxes yield compared with untreated plants.
Karkade treated without adding MWCNT and 10 ml/L Delfan plus produced 13 bioactive constituents (Table 5). Acetic acid was identified as a major chemical constituent depending on the GC-MS chromatogram that showed 13 peaks, while thujone gave the highest peak ( Figure 2).
Result in Table 6 explained that karkade treated with 500 mg/L MWCNT and without adding Delfan plus produced 12 bioactive constituents. Depending on the GC-MS chromatogram in Figure 3, 1,2-benzenedicarboxylic acid, mono (2-ethylhexyl) ester gave the highest relative area and peak compared to other compounds.

Discussions
On the basis of previous results, the reasons for significant increasing growth indicators by adding factor are due to the fact that multiwalled carbon nanotubes (MWCNT) have sizes ranging from 8 to 15 nm (Table 1) and a large amount of surface area in relation to mass and thus have a high ability to penetrate the walls of plant cells and act as a smart transport system in the plant as well as multiple walls increasing the surface area of the mass; these results are consistent with Canas et al. [11] on the carrot, cabbage, lettuce, cucumber, and onion, Khodakovskaya et al. [12] on the tomato, and Heydari [13] on the anthurium.
Journal of Botany 5   Extensive research has shown that the penetration of MWCNT into the plant cells can bring about changes in metabolic activity, leading to an increase in biomass. The tubular nanoparticle is easy to transport inside the plant. MWCNT increased water uptake through changes in the lipid formation, rigidity, and permeability of cell membrane and also enhanced net assimilation of CO 2 . MWCNT might act as regulators for growth or could regulate the marker genes to enhance cell culture growth by increasing cell wall formation, cell divisions, and water transport [14,15]. MWCNT can penetrate cell wall of leaves and induce growth of plants and development (enhancement of shoot length and root length). They also could organize nutrients absorption in the plant such as increased nitrogen, potassium, calcium, and phosphorus while decreasing sodium percent and as a result increasing total chlorophyll [16].
Additionally, MWCNT can be introduced in the plant cell by their attachment to carrier proteins, through aquaporins, using ionic channels, by endocytosis, and through their merging with organic compounds. In the particular case of MWCNT, their entrance is associated mainly with the creation of new pores in the cells [17]. The characteristics are unique of MWCNT (nanoparticle size, ability to penetrate cells, high biochemical reactivity, and rapid distribution inside plant system) and make them an attractive tool for crop management techniques. In this respect, specific types of nanoparticles in low doses have not been found to be harmful to plants but instead are capable of activating specific physiological processes, promoting photosynthesis and nitrogen metabolism, and therefore improving the growth of the plants [18]. They also play a key role in cell wall reinforcement during plant development, cell elongation,    TIC  1900000  1850000  1800000  1750000  1700000  1650000  1600000  1550000  1500000  1450000  1400000  1350000  1300000  1250000  1200000  1150000  1100000  1050000  1000000  950000  900000  850000  800000  750000  700000  650000  600000  550000  500000  450000  400000  350000  300000  250000  200000  150000  100000   high concentration of multiwalled carbon nanotubes to plant probably causes stress to the plant.

Conclusions
Data from results suggest that experimental factors have an important role in causing quantitative and qualitative variations of their medically active ingredients compared with untreated plants. Combination treatment of 1000 mg/L multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan plus) caused an increase in all studied parameters of karkade.

Conflicts of Interest
The author declares that they have no conflicts of interest.