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

1. 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 ofkarkade 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.

2. 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].

2.1. Statistical Analysis. Data analysis was carried out using Randomized Complete Blocks Design (RCBD) in a factorial arrangement (3 x 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].

3. 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.

The GC-MS analysis of water extract from calyxes of karkade showed that untreated plants contain 4 bioactive constituents: 3,7,11,15-tetramethyl-2-hexadecen-1-ol; 1,1-bicyclohexyl, 2-(2-methylpropyl)-trans; 3-buten-2-one,4(2-hydroxy-2,6,6-trimethylcyclohexyl); and 1,2-benzenedicarboxylic acid, diisooctyl ester (Table 4). 3-Buten-2-one, 4-(2-hydroxy-2,6,6-trimethylcyclohexyl) was identified as a major chemical constituent and gave the highest peak and relative area depending on the GC-MS chromatogram that showed 4 peaks (Figure 1).

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.

The treatment of karkade with a combination of 500 mg/L MWCNT and 10 ml/L Delfan plus showed 47 bioactive constituents (Table 7). Menthol, 1-(butyn-3-one-1-yl)-(1R,2S,5R) showed the highest relative area and peak compared to other compounds depending on the GC-MS chromatogram (Figure 4).

Karkade treated with 1000 mg/L MWCNT and without adding Delfan plus produced 41 bioactive constituents (Table 8). 1,2-Benzenedicarboxylic acid, diisooctyl ester gave the highest relative area and peak compared to other compounds depending on the GC-MS chromatogram (Figure 5). Finally, karkade treated with a combination of 100 mg/L MWCNT and 10 ml/L Delfan plus showed 60 bioactive constituents (Table 9). Depending on the GC-MS chromatogram in Figure 6, Naphthalenol,decahydro-1,4a dimethyl-7-(1-methylidene)-,[1R(1.alpha.,4a.beta.,8a.alpha.)] showed the highest relative area and peak compared to other compounds.

4. 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.

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 C02. 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, reproduction, and photosynthesis and in response to external signals regulate water channels (aquaporins). There are many reports indicating that aquaporins are central components in plant water relations and are crucial for root water uptake and are a limiting factor for plant growth [19].

Meanwhile, biostimulators (Delfan plus) also caused a significant increase in all studied parameters because they contain free amino acids, total nitrogen, organic matter, and organic carbon (Table 2). These results were in agreement with Saeed et al. [20] on soybean, El-Zohiri and Asfour [21] on potato, and Abo Sedera et al. [22] on strawberry.

Biostimulators act as source of plant growth hormones like gibberellins that could directly or indirectly impact the physiological activities of plant growth and development. The division in the plant with addition of biostimulators leads to increasing the rate of photosynthesis of production of secondary metabolic compounds of plants, particularly in the case of abiotic stress.

Application of exogenous amino acid improves plant growth by increasing the concentration of photosynthetic pigments (ALA is a common precursor to tetrapyrrole) [23, 24], as well as antioxidative enzyme activities [25]. And it affects processes involved in plant nitrogen metabolism (Iwai et al. [26]). Also, amino acids in biostimulant have positive effects on plant growth and yield and significantly relieve the injuries caused by abiotic stresses [8]; the addition of high concentration of multiwalled carbon nanotubes to plant probably causes stress to the plant.

5. 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.

https://doi.org/ 10.1155/2018/9097363

Conflicts of Interest

The author declares that they have no conflicts of interest.

References

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Layth Sareea Al-Rekaby

Department of Biology, College of Science, University of Al-Qadisiyah, Qadisiyyah, Iraq

Correspondence should be addressed to Layth Sareea Al-Rekaby; layth.sareea@qu.edu.iq

Received 22 August 2017; Revised 14 December 2017; Accepted 19 December 2017; Published 11 February 2018

Academic Editor: Shamsul Hayat

Caption: FIGURE 1: GC-MS chromatogram of water extract of calyxes of karkade (Hibiscus sabdariffa L.) from combination treatment without multiwalled carbon nanotubes and biostimulators (Delfan plus).

Caption: FIGURE 2: GC-MS chromatogram of water extract of calyxes of karkade (Hibiscus sabdariffa L.) from combination treatment without multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan plus).

Caption: FIGURE 3: GC-MS chromatogram of water extract of calyxes of karkade (Hibiscus sabdariffa L.) from combination treatment of 500 mg/L multiwalled carbon nanotubes and without biostimulators (Delfan plus).

Caption: FIGURE 4: GC-MS chromatogram of water extract of calyxes of karkade (Hibiscus sabdariffa L.) from combination treatment of 500 mg/L multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan plus).

Caption: Figure 5: GC-MS chromatogram of water extract of calyxes of karkade (Hibiscus sabdariffa L.) from combination treatment of 1000 mg/L multiwalled carbon nanotubes and without biostimulators (Delfan plus).

Caption: Figure 6: GC-MS chromatogram of water extract of calyxes of Hibiscus sabdariffa L. from combination treatment of 1000 mg/L multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan plus).
TABLE 1: Characteristics of multiwalled carbon nanotubes
(MWCNT).

Aspect                         Powder

Colour                          Black
Length                     10-50 [micro]m
Outer diameter                 8-15 nm
Inside diameter                3-5 nm
Specific surface area      233 [m.sup.2]/g
Purity                         >95 wt%
True density              ~2.1g/[cm.sup.3]
Bulk density              0.15 g/[cm.sup.3]
Ash                           <1.5 wt%
Electrical conductivity       >100 s/cm

TABLE 2: Characteristics of biostimulators (Delfan plus).

Physical-chemical             Chemical analysis
characteristics

Aspect     Liquid     Free amino acids    24.00%
Colour     Brown     Total nitrogen (N)   9.00%
Density   1.2 g/cc     Organic matter     37.00%
pH          7.2        Organic carbon     23.00%

TABLE 3: Significant effect of multiwalled carbon nanotubes and
biostimulators (Delfan plus) in the studied characteristics of
Hibiscus sabdariffa L.

Multiwalled        Biostimulators   Length of (cm)
carbon nanotubes   (Delfan plus)
concentration      concentration    Shoot   Root
(mg/L)                 (ml/L)

0                        0          51.14   18.13
                         10         57.44   22.43
500                      0          52.13   23.54
                         10         64.81   26.22
1000                     0          59.73   28.73
                         10         73.55   33.41
RLSD at 0.05                        9.55    2.47

Multiwalled        Number of (per plant)        Chlorophyll
carbon nanotubes                                  content
concentration      Leaves   Branches   Fruits     (SPAD)
(mg/L)

0                  15.63      6.62     36.22       51.55
                   23.33     11.42     44.13       53.42
500                25.31      8.32     57.81       54.82
                   33.14     14.52     69.11       56.19
1000               29.46     10.74     77.54       57.22
                   44.52     17.46     92.83       59.78
RLSD at 0.05        8.69      4.63     10.61       2.33

Multiwalled          DW of       Number of
carbon nanotubes     calyx       bioactive
concentration        yield      constituents
(mg/L)             (gm/fruit)   (per plant)

0                    24.52           4
                     29.72           13
500                  31.61           12
                     36.62           47
1000                 40.41           41
                     47.43           60
RLSD at 0.05          5.51          7.97

Table 4: Bioactive constituents of water extract of calyxes of
karkade (Hibiscus sabdariffa L.) from combination treatment without
multiwalled carbon nanotubes and biostimulators (Delfan plus).

                       Peak report TIC

Peak   R. time    Area    Area%    Height   Height%

1       12.62    93268    10.28    19921     24.88
2       13.56    70006     7.72    12624     15.77
3       18.57    689870   76.07    35869     44.80
4       20.42    53697     5.92    11659     14.56
                 906841   100.00   80073    100.00

                           Peak report TIC

Peak                             Name

1               3,7,11,15-Tetramethyl-2-hexadecen-1-ol
2           1,1'-Bicyclohexyl, 2-(2-methylpropyl)-, trans-
3      3-Buten-2-one, 4-(2-hydroxy-2,6,6-trimethylcyclohexyl)-
4           1,2-Benzenedicarboxylic acid, diisooctyl ester

TABLE 5: Bioactive constituents of water extract of calyxes of
karkade (Hibiscus sabdariffa L.) from combination treatment without
multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan
plus).

                 Peak report TIC

Peak   R. time    Area     Area%    Height   Height%

1       5.87     924451    28.07    44787     9.86
2       6.98     234129     7.11    18401     4.05
3       9.39     264901     8.04    23849     5.25
4       10.45     66366     2.01    13395     2.95
5       11.63     58361     1.77    16685     3.67
6       12.58    237929     7.22    78034     17.18
7       13.31     45794     1.39    14885     3.28
8       13.60    200938     6.10    28416     6.26
9       16.87    187103     5.68    65937     14.52
10      17.22     96494     2.93    17373     3.82
11      18.00     72451     2.20    23296     5.13

12      18.42    487748    14.81    27530     6.06
13      22.88    416944    12.66    81663     17.98
                 3293609   100.00   454251   100.00

                           Peak report TIC

Peak                             Name

1                            Acetic acid
2                              Camphor
3         Bicyclo[2.2.1]heptane, 2-methoxy-1,7,7-trimethyl-
4                  2-Butenoic acid, 2-methyl-, (Z)-
5                           1-Hexadecanol
6               3,7,11,15-Tetramethyl-2-hexadecen-1-ol
7             1,3-Cyclopentadiene, 5,5-dimethyl-2-ethyl-
8       Diazoacetic acid, 2-isopropyl-5-methylcyclohexyl ester
9               3,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-
10      3-0xabicyclo[4.1.0]heptan-2-one, 4,4,7,7-tetramethyl-
11              12-0xabicyclo[9.1.0]dodeca-3,7-diene,
             1,5,5,8-tetramethyl-, [1R-(1R*,3E,7E,11R*)]-
12     3-Buten-2-one, 4-(2-hydroxy-2,6,6-trimethylcyclohexyl)-
13                             Thujone

TABLE 6: Bioactive constituents of water extract of calyxes of
karkade (Hibiscus sabdariffa L.) from combination treatment of 500
mg/L multiwalled carbon nanotubes and without biostimulators (Delfan
plus).

                           Peak report TIC

Peak   R. time    Area     Area%    Height   Height%

1       5.10     786921    11.31    30384     4.41
2       14.29    1861443   26.75    84961     12.34

3       15.90    210700     3.03    35279     5.12
4       16.94    349173     5.02    37447     5.44
5       17.63     11414     0.16     4964     0.72
6       17.73     48075     0.69    15669     2.28
7       17.81     41981     0.60    15121     2.20
8       19.17    820233    11.79    115136    16.72
9       20.48    2197954   31.59    278588    40.46

10      21.56    141100     2.03    16363     2.38
11      22.29    127819     1.84    23936     3.48
12      23.36    360897     5.19    30782     4.47
                 6957710   100.00   688630   100.00

                Peak report TIC

Peak                  Name

1       2-Pentanone, 4-hydroxy-4-methyl-
2      2(3H)-Benzofuranone, hexahydro-3a,
               7a-dimethyl-, cis-
3               Tritetracontane
4        Docosyl pentafluoropropionate
5               Tetratriacontane
6              Tetratetracontane
7               Tritetracontane
8               Tetratriacontane
9        1,2-Benzenedicarboxylic acid,
            mono(2-ethylhexyl) ester
10          Eicosane, 2-cyclohexyl-
11               1-Heneicosanol
12        cis-13,16-Docasadienoic acid

TABLE 7: Bioactive constituents of water extract of calyxes of
karkade (Hibiscus sabdariffa L.) from combination treatment of 500
mg/L multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan
plus).

                         Peak report TIC

Peak   R. time     Area     Area%    Height    Height%

1       8.82      141226     0.94     14453     0.41
2       9.46      162535     1.09     20610     0.58

3       9.93      248143     1.66     19212     0.54
4       10.48     481302     3.22     80512     2.28
5       11.60     129429     0.87     20711     0.59

6       12.35     108383     0.72     28348     0.80
7       12.64     269674     1.80     79065     2.24
8       13.00     244025     1.63     86620     2.45
9       13.33     293411     1.96     63445     1.80
10      13.57     194491     1.30     30385     0.86
11      13.83     67118      0.45     19221     0.54

12      14.66     45902      0.31     17959     0.51
13      14.76     93078      0.62     34590     0.98

14      14.83     175845     1.18     81537     2.31
15      15.06     275082     1.84     28540     0.81
16      15.22     411108     2.75    109720     3.10
17      15.68     400775     2.68     89433     2.53

18      15.83     83819      0.56     31391     0.89
19      16.04     141274     0.94     67137     1.90

20      16.09     351866     2.35    191370     5.42
21      16.36     116915     0.78     48041     1.36
22      16.44     53494      0.36     20051     0.57
23      16.78     159689     1.07     61966     1.75
24      16.88     78982      0.53     33741     0.95

25      16.96     111111     0.74     40230     1.14
26      17.12     209656     1.40     76583     2.17
27      17.30     58891      0.39     24040     0.68
28      17.58     67358      0.45     18877     0.53
29      17.78     63878      0.43     21067     0.60
30      18.00     309133     2.07    120882     3.42
31      18.58     48232      0.32     21779     0.62
32      18.66     149865     1.00     64467     1.82
33      18.94     181596     1.21     51056     1.44
34      19.37     244278     1.63     81298     2.30
35      19.49    1286488     8.60    423334     11.98
36      19.65     186885     1.25     53306     1.51
37      19.82     54252      0.36     18932     0.54
38      20.13     68098      0.46     24263     0.69
39      20.44     971615     6.50     98698     2.79
40      20.62     122710     0.82     32654     0.92
41      20.82     177718     1.19     43055     1.22

42      20.96     885998     5.92    264130     7.47

43      21.54     753783     5.04     95420     2.70
44      22.20     370247     2.48     48444     1.37
45      22.88     304991     2.04     43927     1.24
46      23.13     252921     1.69     27372     0.77

47      23.72    3349386    22.39    561901     15.90
                 14956656   100.00   3533773   100.00

                          Peak report TIC

Peak                           Name

1                             Borneol
2              1H-Benzocycloheptene, 2,4a,5,6,7,8,9,
            9a-octahydro-3,5,5-trimethyl-9-methylene-,
                            (4aS-cis)-
3                2-Butenoic acid, 2-methyl-, (E)-
4                      Dodecan-1-yl acetate
5            Cyclopropanecarboxylic acid, 1-hydroxy-,
               (2,6-di-t-butyl-4-methylphenyl) ester
6             3,7,11,15-Tetramethyl-2-hexadecen-1-ol
7             3,7,11,15-Tetramethyl-2-hexadecen-1-ol
8             3,7,11,15-Tetramethyl-2-hexadecen-1-ol
9                         (-)-Spathulenol
10              2-Pentadecanone, 6,10,14-trimethyl-
11            2-Naphthalenemethanol, 1,2,3,4,4a,5,6,
           7-octahydro-a,a,4a,8-tetramethyl-, (2R-cis)-
12                         Veridiflorol
13            2-Naphthalenemethanol, 2,3,4,4a,5,6,7,
                8-octahydro-a,a,4a,8-tetramethyl-,
                       [2R-(2.alpha.,4a.be)]
14                Hexadecanoic acid, ethyl ester
15                Hexadecanoic acid, ethyl ester
16           1-Hexadecen-3-ol, 3,5,11,15-tetramethyl-
17             Tricyclo[5.2.2.0(1,6)]undecan-3-ol,
                   2-methylene-6,8,8-trimethyl-
18                    Limonen-6-ol, pivalate
19                  2(4H)-Benzofuranone, 5,6,7,
                  7a-tetrahydro-4,4,7a-trimethyl-
20                   Longipinocarveol, trans-
21                   Longipinocarveol, trans-
22         10-Methyltricyclo[4.3.1.1(2,5)]undecan-10-ol
23           Succinic acid, ethyl non-5-yn-3-yl ester
24            Bicyclo[2.2.1]heptan-2-ol, 4-chloro-1,
                       7,7-trimethyl-, exo-
25                        Dodecanoic acid
26            9,12-Octadecadienoic acid, ethyl ester
27            9,12,15-Octadecatrienoic acid, (Z,Z,Z)-
28                            Phytol
29        1,2-Benzenedicarboxylic acid, diisooctyl ester
30         4-(2,2-Dimethyl-6-methylenecyclohexyl)butanal
31            Hexadecanoic acid, 2-methylpropyl ester
32                  Z,Z-8,10-Hexadecadien-1-ol
33                      n-Hexadecanoic acid
34                        Heptanoic acid
35                      Citronellyl tiglate
36          Bicyclo[2.2.1]heptan-2-ol, 2,3,3-trimethyl-
37                       4-Methyldocosane
38                          1-Eicosene
39       1-Cyclohexanone, 2-methyl-2-(3-methyl-2-oxobutyl)
40     3-Buten-2-ol, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-
41           (1S-(1Alpha,2alpha,4beta))-1-isopropenyl-
                   4-methyl-1,2-cyclohexanediol
42                 2H-Indeno[1,2-b]furan-2-one,
             3,3a,4,5,6,7,8,8b-octahydro-8,8-dimethyl
43       1-(3-Methoxymethyl-2,5,6-trimethylphenyl)ethanone
44            Fumaric acid, ethyl 2-methylallyl ester
45                      7-Hexadecenal, (Z)
46                Cyclopropanemethanol,. alpha.,
               2-dimethyl-2-(4-methyl-3-pentenyl)-,
                     [1.alpha.(R*),2.alpha.]-
47         Menthol, 1'-(butyn-3-one-1-yl)-, (1R,2S,5R)-

TABLE 8: Bioactive constituents of water extract of calyxes of
karkade (Hibiscus sabdariffa L.) from combination treatment of 1000
mg/L multiwalled carbon nanotubes and without biostimulators (Delfan
plus).

                 Peak report TIC

Peak   R. time     Area     Area%    Height    Height%

1       7.02      765601     2.17     56572     0.86
2       8.74      82130      0.23     15029     0.23
3       8.87      81976      0.23     10541     0.16
4       9.38      520061     1.47     49940     0.76
5       9.87      32654      0.09     10191     0.16
6       9.91       9809      0.03     5883      0.09
7       10.44    1145521     3.24    132334     2.01
8       11.01     145689     0.41     24839     0.38
9       11.29     78677      0.22     17466     0.27
10      11.66     64866      0.18     19188     0.29
11      12.35     39374      0.11     14212     0.22
12      12.69     106924     0.30     11102     0.17
13      13.30     356944     1.01     73595     1.12
14      13.56     91558      0.26     33046     0.50
15      13.82     291034     0.82     89776     1.37
16      14.66     168921     0.48     78432     1.19
17      14.75     79761      0.23     32959     0.50

18      14.83     408734     1.16    176706     2.69
19      15.09     467380     1.32     68319     1.04
20      15.26     319379     0.90     83584     1.27
21      15.67     32473      0.09     18890     0.29
22      16.05     318485     0.90    143648     2.19
23      16.09     300889     0.85    133271     2.03
24      16.44     281577     0.80     65032     0.99

25      16.60     473246     1.34    223525     3.40
26      16.79     283275     0.80    117213     1.78
27      17.49     92528      0.26     39301     0.60
28      18.00     293141     0.83    105445     1.60
29      18.19     136422     0.39     55398     0.84
30      18.31     447480     1.27    149416     2.27
31      18.51    2937254     8.31    1043306    15.88
32      18.80     151566     0.43     54379     0.83

33      19.57    5322093    15.06    604327     9.20
34      20.75    11971493   33.88    1173046    17.85
35      20.89    1963448     5.56    620603     9.45
36      20.97     441729     1.25    151709     2.31
37      21.66     798268     2.26     78439     1.19
38      22.25     84015      0.24     25211     0.38
39      22.34     448534     1.27     81446     1.24
40      22.90    2376704     6.73    483543     7.36

41      23.70     925582     2.62    199546     3.04
                 35337195   100.00   6570408   100.00

                         Peak report TIC

Peak                          Name

1      Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-
2                   Pentanoic acid, 4-methyl-
3        1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-
4                          Isoborneol
5                         Caryophyllene
6                     Geranyl ethyl ether 2
7               2-Butenoic acid, 2-methyl-, (E)-
8                  Dodecane, 2,6,11-trimethyl-
9                         Hexanoic acid
10                       1-Heneicosanol
11             Heptadecane, 2,6,10,15-tetramethyl-
12         2-Butanone, 4-(2,2,6-trimethylcyclohexyl)-
13                     Caryophyllene oxide
14                    Octadecane, 1-chloro-
15      1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-
16                         Heneicosane
17                1H-Cycloprop[e]azulen-7-ol,
             decahydro-1,1,7-trimethyl-4-methylene-,
       [1ar-(1a.alpha.,4a.alpha.,7.beta.,7a.beta.,7b.alpha.)]
18             2-Pentadecanone, 6,10,14-trimethyl-
19                       1-Heneicosanol
20                         Caprolactam
21                     2-Bromotetradecane
22                      Selina-6-en-4-ol
23               Hexadecanoic acid, ethyl ester
24             1H-3a,7-Methanoazulene-6-methanol,
            2,3,4,7,8,8a-hexahydro-3,8,8-trimethyl-,
           [3R-(3.alpha.,3a.beta.,7.beta.,8a.alpha.)]
25                         Heneicosane
26        8-Hydroxy-2,2,8-trimethyldeca-5,9-dien-3-one
27             Hexadecane, 2,6,11,15-tetramethyl-
28                       Eicosanoic acid
29                   2-Decene, 2,4-dimethyl-
30      1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-
31                    Eicosane, 10-methyl-
32               2,6-Octadienal, 2,6-dimethyl-8-
                  (tetrahydro-2H-2-pyranyloxy)
33                       Hentriacontane
34       1,2-Benzenedicarboxylic acid, diisooctyl ester
35                         Heneicosane
36         Oxalic acid, 2-ethylhexyl pentadecyl ester
37                cis-13,16-Docasadienoic acid
38     (2,2,6-Trimethyl-bicyclo[4.1.0]hept-1-yl)-methanol
39                       Tritetracontane
40                   Cyclopropanemethanol,.
           alpha.,2-dimethyl-2-(4-methyl-3-pentenyl)-,
                    [1.alpha.(R*),2.alpha.]-
41                     Pentadecanoic acid

TABLE 9: Bioactive constituents of water extract of calyxes of
Hibiscus sabdariffa L. from combination treatment of 1000 mg/L
multiwalled carbon nanotubes and 10 ml/L biostimulators (Delfan
plus).

                 Peak Report TIC

Peak   R. time     Area     Area%    Height    Height%

1       5.98      179688     0.89     14717     0.29
2       6.53      206765     1.03     16890     0.33
3       6.99      477865     2.38     39661     0.77
4       7.75      17402      0.09     8436      0.16
5       7.99      277810     1.38     25340     0.49
6       8.72      58849      0.29     11308     0.22
7       8.89      61164      0.30     11322     0.22
8       9.38      478340     2.38     65463     1.27
9       9.84      13698      0.07     7413      0.14

10      9.88      11189      0.06     8044      0.16

11      10.48     861378     4.28    116784     2.27
12      10.99     54684      0.27     15680     0.31
13      11.29     23403      0.12     13933     0.27
14      11.40     96098      0.48     29717     0.58
15      11.62     96495      0.48     30081     0.59
16      12.34     43727      0.22     14425     0.28
17      12.58     72201      0.36     17495     0.34
18      12.96     95214      0.47     31350     0.61
19      13.30     413748     2.06    114634     2.23

20      13.56     58576      0.29     18742     0.37
21      13.81     333618     1.66    122979     2.40
22      13.94     257018     1.28     76005     1.48
23      14.00     27371      0.14     18033     0.35
24      14.15     101642     0.51     25064     0.49
25      14.24     103868     0.52     28501     0.56
26      14.36     65809      0.33     24124     0.47
27      14.66     219672     1.09    107246     2.09
28      14.74     136624     0.68     55418     1.08

29      14.83     943433     4.69    399599     7.78
30      15.02     72288      0.36     29358     0.57
31      15.11     221117     1.10     85066     1.66

32      15.22     334667     1.66     83563     1.63

33      15.34     21275      0.11     13398     0.26
34      15.82     179493     0.89     59327     1.16

35      16.04    2866546    14.25    855621     16.66

36      16.41     159687     0.79     39983     0.78

37      16.59     474369     2.36    220498     4.29
38      16.78     432738     2.15    180735     3.52
39      16.96     250742     1.25     76006     1.48
40      17.12     144462     0.72     59742     1.16
41      17.23     116967     0.58     29430     0.57
42      17.39     149266     0.74     44782     0.87
43      17.57     118813     0.59     37221     0.72
44      17.91     52764      0.26     16286     0.32
45      18.00     278732     1.39    107308     2.09
46      18.18     203495     1.01     68303     1.33
47      18.46     799323     3.97    240431     4.68
48      18.65     168921     0.84     74823     1.46
49      19.46     224542     1.12     39309     0.77
50      19.92     156623     0.78     28959     0.56

51      20.21     144167     0.72     40315     0.79
52      20.31     219523     1.09     59841     1.17
53      20.50     49486      0.25     17568     0.34
54      20.60     195477     0.97     54141     1.05
55      20.80     253010     1.26     76165     1.48
56      20.94     441843     2.20    129566     2.52
57      21.05     123783     0.62     38025     0.74
58      21.83    1095808     5.45     63024     1.23
59      22.88    1803739     8.97    328164     6.39
60      23.70    2578313    12.82    469027     9.14
                 20119328   100.00   5134389   100.00

                            Peak Report TIC

Peak                             Name

1              1-[t-Butyl]-3-[2-hydroxyethyl]-2-thiourea
2       1,3,3-Trimethylcyclohex-1-ene-4-carboxaldehyde, (+,-)-
3         Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-
4              2-Octenoic acid, 7-hydroxy-, ethyl ester
5                    2-Cyclohexen-1-one, 3-methyl-
6                      Decanoic acid, 3-methyl-
7        1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-, (Z)-
8                               Borneol
9        1H-Cycloprop[e]azulene, decahydro-1,1,7-trimethyl-4-
       methylene-, [1aR-(1a.alpha.,4a.alpha.,7.alpha.,7a.beta.,
                             7b. alpha.)]
10          Spiro[cyclopropane-1,8'(1H')[3a.6]methano[3ah]
              cyclopentacycloocten]-10'-one, octahydro-,
                            (3'as,6'R,9'ar)
11                 2-Butenoic acid, 2-methyl-, (E)-
12                         Decane, 2-methyl-
13                          Heptanoic acid
14          2-Hexanol, 3,3,5-trimethyl-2-(3-methylphenyl)-
15                          n-Nonadecanol-1
16                         Decane, 2-methyl-
17                    E-6-Octadecen-1-ol acetate
18                            1-Dodecanol
19           Tricyclo[3.1.0.0(2,4)]hexane, 3,6-diethyl-3,
                          6-dimethyl-, trans-
20            2-Cyclohexene-1-methanol, 2,6,6-trimethyl-
21                         Nerolidyl acetate
22       2,3-Dipropyl-cyclopropanecarboxylic acid, ethyl ester
23                  Tetradecanoic acid, ethyl ester
24                  10s,11s-Himachala-3(12),4-diene
25                2H-Pyran-2-one, 4-hydroxy-6-methyl-
26                          Terpin Hydrate
27                        2-Bromotetradecane
28      1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-
           4-methylene-, [1ar-(1a.alpha.,4a.alpha.,7.beta.,
                         7a.beta.,7b.alpha.)]
29                2-Pentadecanone, 6,10,14-trimethyl-
30        2-Piperidinone, 1-(3,4,5,6-tetrahydro-2-pyridinyl)-
31                    7-Oxabicyclo[4.1.0]heptane,
             2,2,6-trimethyl-1-(3-methyl-1,3-butadienyl)-
                             5-methylene-
32               2H-Cyclopentacyclooctene, 4,5,6,7,8,
                   9-hexahydro-1,2,2,3-tetramethyl-
33                     Heptanoic acid, anhydride
34              1H-Cycloprop[e]azulene, decahydro-1,1,
                       7-trimethyl-4-methylene-
35            1-Naphthalenol, decahydro-1,4a-dimethyl-7-
                        (1-methylethylidene)-,
                  [1R-(1.alpha.,4a.beta.,8a.alpha.)]-
36                9-Isopropyl-1-methyl-2-methylene-5-
                   oxatricyclo[5.4.0.0(3,8)]undecane
37                             Tricosane
38                      Limonen-6-ol, pivalate
39                          (-)-Spathulenol
40                     Longipinocarveol, trans-
41           1-Cyclohexyl-1-(2-methylenecyclohexyl)ethanol
42                        Cyclohexane, butyl-
43                  (1-Ethylbuta-1,3-dienyl)benzene
44                  Benzeneethanol,. alpha.-ethyl-
45                          Dodecanoic acid
46          But-2-enoic acid, amide, 3-methyl-N-methallyl-
47                            Heptacosane
48              9,12-Octadecadienoic acid, ethyl ester
49                        Phytol Acetic acid,
50       3-hydroxy-6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,
                   8a-octahydronaphthalen-2-yl ester
51                    Oxacycloheptadec-8-en-2-one
52                        Tetradecanoic acid
53           1,3-Dioxolane, 4,4,5-trimethyl-2-pentadecyl-
54              Fumaric acid, ethyl 2-methylallyl ester
55                         Tetratetracontane
56            Oxalic acid, 2-ethylhexyl pentadecyl ester
57              Cyclohexane, 1,5-dimethyl-2,3-divinyl-
58                           1-Octacosanol
59              Cyclohexan-1-ethanol, 1-hydroxymethyl-
60                        n-Hexadecanoic acid
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Title Annotation:Research Article
Author:Al-Rekaby, Layth Sareea
Publication:Journal of Botany
Article Type:Report
Date:Jan 1, 2018
Words:5973
Previous Article:Experimental Treatment with a Hypomethylating Agent Alters Life History Traits and Fitness in Brassica rapa.
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