Journal of Botany
Volume 2018 (2018), Article ID 9097363, 11 pages
https://doi.org/10.1155/2018/9097363

Research Article

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

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

Copyright © 2018 Layth Sareea Al-Rekaby. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

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.

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 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.

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.

Table 1: Characteristics of multiwalled carbon nanotubes (MWCNT).

Aspect
Powder

Colour
Black

Length
10–50 µm

Outer diameter
8–15 nm

Inside diameter
3–5 nm

Specific surface area
233 m²/g

Purity
>95 wt%

True density
~2.1 g/cm³

Bulk density
0.15 g/cm³

Ash
<1.5 wt%

Electrical conductivity
>100 s/cm

Table 2: Characteristics of biostimulators (Delfan plus).

Physical-chemical characteristics
Chemical analysis

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%

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 × 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.

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

Multiwalled carbon nanotubes concentration (mg/L)
Biostimulators (Delfan plus) concentration (ml/L)
Length of (cm)
Number of (per plant)
Chlorophyll content (SPAD)
DW of calyx yield (gm/fruit)
Number of bioactive constituents (per plant)

Shoot
Root
Leaves
Branches
Fruits

0
0
51.14
18.13
15.63
6.62
36.22
51.55
24.52
4

10
57.44
22.43
23.33
11.42
44.13
53.42
29.72
13

500
0
52.13
23.54
25.31
8.32
57.81
54.82
31.61
12

10
64.81
26.22
33.14
14.52
69.11
56.19
36.62
47

1000
0
59.73
28.73
29.46
10.74
77.54
57.22
40.41
41

10
73.55
33.41
44.52
17.46
92.83
59.78
47.43
60

RLSD at 0.05
9.55
2.47
8.69
4.63
10.61
2.33
5.51
7.97

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).

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%
Name

1
12.62
93268
10.28
19921
24.88
3,7,11,15-Tetramethyl-2-hexadecen-1-ol

2
13.56
70006
7.72
12624
15.77
1,1′-Bicyclohexyl, 2-(2-methylpropyl)-, trans-

3
18.57
689870
76.07
35869
44.80
3-Buten-2-one, 4-(2-hydroxy-2,6,6-trimethylcyclohexyl)-

4
20.42
53697
5.92
11659
14.56
1,2-Benzenedicarboxylic acid, diisooctyl ester

906841
100.00
80073
100.00

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).

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).

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%
Name

1
5.87
924451
28.07
44787
9.86
Acetic acid

2
6.98
234129
7.11
18401
4.05
Camphor

3
9.39
264901
8.04
23849
5.25
Bicyclo[2.2.1]heptane, 2-methoxy-1,7,7-trimethyl-

4
10.45
66366
2.01
13395
2.95
2-Butenoic acid, 2-methyl-, (Z)-

5
11.63
58361
1.77
16685
3.67
1-Hexadecanol

6
12.58
237929
7.22
78034
17.18
3,7,11,15-Tetramethyl-2-hexadecen-1-ol

7
13.31
45794
1.39
14885
3.28
1,3-Cyclopentadiene, 5,5-dimethyl-2-ethyl-

8
13.60
200938
6.10
28416
6.26
Diazoacetic acid, 2-isopropyl-5-methylcyclohexyl ester

9
16.87
187103
5.68
65937
14.52
3,6-Octadien-1-ol, 3,7-dimethyl-, (Z)-

10
17.22
96494
2.93
17373
3.82
3-Oxabicyclo[4.1.0]heptan-2-one, 4,4,7,7-tetramethyl-

11
18.00
72451
2.20
23296
5.13
12-Oxabicyclo[9.1.0]dodeca-3,7-diene, 1,5,5,8-tetramethyl-, [1R-(1R,3E,7E,11R)]-

12
18.42
487748
14.81
27530
6.06
3-Buten-2-one, 4-(2-hydroxy-2,6,6-trimethylcyclohexyl)-

13
22.88
416944
12.66
81663
17.98
Thujone

3293609
100.00
454251
100.00

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).

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.

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%
Name

1
5.10
786921
11.31
30384
4.41
2-Pentanone, 4-hydroxy-4-methyl-

2
14.29
1861443
26.75
84961
12.34
2(3H)-Benzofuranone, hexahydro-3a,7a-dimethyl-, cis-

3
15.90
210700
3.03
35279
5.12
Tritetracontane

4
16.94
349173
5.02
37447
5.44
Docosyl pentafluoropropionate

5
17.63
11414
0.16
4964
0.72
Tetratriacontane

6
17.73
48075
0.69
15669
2.28
Tetratetracontane

7
17.81
41981
0.60
15121
2.20
Tritetracontane

8
19.17
820233
11.79
115136
16.72
Tetratriacontane

9
20.48
2197954
31.59
278588
40.46
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester

10
21.56
141100
2.03
16363
2.38
Eicosane, 2-cyclohexyl-

11
22.29
127819
1.84
23936
3.48
1-Heneicosanol

12
23.36
360897
5.19
30782
4.47
cis-13,16-Docasadienoic acid

6957710
100.00
688630
100.00

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).

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).

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%
Name

1
8.82
141226
0.94
14453
0.41
Borneol

2
9.46
162535
1.09
20610
0.58
1H-Benzocycloheptene, 2,4a,5,6,7,8,9,9a-octahydro-3,5,5-trimethyl-9-methylene-, (4aS-cis)-

3
9.93
248143
1.66
19212
0.54
2-Butenoic acid, 2-methyl-, (E)-

4
10.48
481302
3.22
80512
2.28
Dodecan-1-yl acetate

5
11.60
129429
0.87
20711
0.59
Cyclopropanecarboxylic acid, 1-hydroxy-, (2,6-di-t-butyl-4-methylphenyl) ester

6
12.35
108383
0.72
28348
0.80
3,7,11,15-Tetramethyl-2-hexadecen-1-ol

7
12.64
269674
1.80
79065
2.24
3,7,11,15-Tetramethyl-2-hexadecen-1-ol

8
13.00
244025
1.63
86620
2.45
3,7,11,15-Tetramethyl-2-hexadecen-1-ol

9
13.33
293411
1.96
63445
1.80
(−)-Spathulenol

10
13.57
194491
1.30
30385
0.86
2-Pentadecanone, 6,10,14-trimethyl-

11
13.83
67118
0.45
19221
0.54
2-Naphthalenemethanol, 1,2,3,4,4a,5,6,7-octahydro-α,α,4a,8-tetramethyl-, (2R-cis)-

12
14.66
45902
0.31
17959
0.51
Veridiflorol

13
14.76
93078
0.62
34590
0.98
2-Naphthalenemethanol, 2,3,4,4a,5,6,7,8-octahydro-α,α,4a,8-tetramethyl-, [2R-(2.alpha.,4a.be)]

14
14.83
175845
1.18
81537
2.31
Hexadecanoic acid, ethyl ester

15
15.06
275082
1.84
28540
0.81
Hexadecanoic acid, ethyl ester

16
15.22
411108
2.75
109720
3.10
1-Hexadecen-3-ol, 3,5,11,15-tetramethyl-

17
15.68
400775
2.68
89433
2.53
Tricyclo[5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8-trimethyl-

18
15.83
83819
0.56
31391
0.89
Limonen-6-ol, pivalate

19
16.04
141274
0.94
67137
1.90
2(4H)-Benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-

20
16.09
351866
2.35
191370
5.42
Longipinocarveol, trans-

21
16.36
116915
0.78
48041
1.36
Longipinocarveol, trans-

22
16.44
53494
0.36
20051
0.57
10-Methyltricyclo[4.3.1.1(2,5)]undecan-10-ol

23
16.78
159689
1.07
61966
1.75
Succinic acid, ethyl non-5-yn-3-yl ester

24
16.88
78982
0.53
33741
0.95
Bicyclo[2.2.1]heptan-2-ol, 4-chloro-1,7,7-trimethyl-, exo-

25
16.96
111111
0.74
40230
1.14
Dodecanoic acid

26
17.12
209656
1.40
76583
2.17
9,12-Octadecadienoic acid, ethyl ester

27
17.30
58891
0.39
24040
0.68
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-

28
17.58
67358
0.45
18877
0.53
Phytol

29
17.78
63878
0.43
21067
0.60
1,2-Benzenedicarboxylic acid, diisooctyl ester

30
18.00
309133
2.07
120882
3.42
4-(2,2-Dimethyl-6-methylenecyclohexyl)butanal

31
18.58
48232
0.32
21779
0.62
Hexadecanoic acid, 2-methylpropyl ester

32
18.66
149865
1.00
64467
1.82
Z,Z-8,10-Hexadecadien-1-ol

33
18.94
181596
1.21
51056
1.44
n-Hexadecanoic acid

34
19.37
244278
1.63
81298
2.30
Heptanoic acid

35
19.49
1286488
8.60
423334
11.98
Citronellyl tiglate

36
19.65
186885
1.25
53306
1.51
Bicyclo[2.2.1]heptan-2-ol, 2,3,3-trimethyl-

37
19.82
54252
0.36
18932
0.54
4-Methyldocosane

38
20.13
68098
0.46
24263
0.69
1-Eicosene

39
20.44
971615
6.50
98698
2.79
1-Cyclohexanone, 2-methyl-2-(3-methyl-2-oxobutyl)

40
20.62
122710
0.82
32654
0.92
3-Buten-2-ol, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-

41
20.82
177718
1.19
43055
1.22
(1S-(1Alpha,2alpha,4beta))-1-isopropenyl-4-methyl-1,2-cyclohexanediol

42
20.96
885998
5.92
264130
7.47
2H-Indeno[1,2-b]furan-2-one, 3,3a,4,5,6,7,8,8b-octahydro-8,8-dimethyl

43
21.54
753783
5.04
95420
2.70
1-(3-Methoxymethyl-2,5,6-trimethylphenyl)ethanone

44
22.20
370247
2.48
48444
1.37
Fumaric acid, ethyl 2-methylallyl ester

45
22.88
304991
2.04
43927
1.24
7-Hexadecenal, (Z)

46
23.13
252921
1.69
27372
0.77
Cyclopropanemethanol,. alpha.,2-dimethyl-2-(4-methyl-3-pentenyl)-, [1.alpha.(R),2.alpha.]-

47
23.72
3349386
22.39
561901
15.90
Menthol, 1′-(butyn-3-one-1-yl)-, (1R,2S,5R)-

14956656
100.00
3533773
100.00

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).

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.

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%
Name

7.02
765601
2.17
56572
0.86
Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-

8.74
82130
0.23
15029
0.23
Pentanoic acid, 4-methyl-

8.87
81976
0.23
10541
0.16
1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-

9.38
520061
1.47
49940
0.76
Isoborneol

9.87
32654
0.09
10191
0.16
Caryophyllene

9.91
9809
0.03
5883
0.09
Geranyl ethyl ether 2

10.44
1145521
3.24
132334
2.01
2-Butenoic acid, 2-methyl-, (E)-

11.01
145689
0.41
24839
0.38
Dodecane, 2,6,11-trimethyl-

11.29
78677
0.22
17466
0.27
Hexanoic acid

11.66
64866
0.18
19188
0.29
1-Heneicosanol

12.35
39374
0.11
14212
0.22
Heptadecane, 2,6,10,15-tetramethyl-

12.69
106924
0.30
11102
0.17
2-Butanone, 4-(2,2,6-trimethylcyclohexyl)-

13.30
356944
1.01
73595
1.12
Caryophyllene oxide

13.56
91558
0.26
33046
0.50
Octadecane, 1-chloro-

13.82
291034
0.82
89776
1.37
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-

14.66
168921
0.48
78432
1.19
Heneicosane

14.75
79761
0.23
32959
0.50
1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene-, [1ar-(1a.alpha.,4a.alpha.,7.beta.,7a.beta.,7b.alpha.)]

14.83
408734
1.16
176706
2.69
2-Pentadecanone, 6,10,14-trimethyl-

15.09
467380
1.32
68319
1.04
1-Heneicosanol

15.26
319379
0.90
83584
1.27
Caprolactam

15.67
32473
0.09
18890
0.29
2-Bromotetradecane

16.05
318485
0.90
143648
2.19
Selina-6-en-4-ol

16.09
300889
0.85
133271
2.03
Hexadecanoic acid, ethyl ester

16.44
281577
0.80
65032
0.99
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.)]

16.60
473246
1.34
223525
3.40
Heneicosane

16.79
283275
0.80
117213
1.78
8-Hydroxy-2,2,8-trimethyldeca-5,9-dien-3-one

17.49
92528
0.26
39301
0.60
Hexadecane, 2,6,11,15-tetramethyl-

18.00
293141
0.83
105445
1.60
Eicosanoic acid

18.19
136422
0.39
55398
0.84
2-Decene, 2,4-dimethyl-

18.31
447480
1.27
149416
2.27
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-

18.51
2937254
8.31
1043306
15.88
Eicosane, 10-methyl-

18.80
151566
0.43
54379
0.83
2,6-Octadienal, 2,6-dimethyl-8-(tetrahydro-2H-2-pyranyloxy)

19.57
5322093
15.06
604327
9.20
Hentriacontane

20.75
11971493
33.88
1173046
17.85
1,2-Benzenedicarboxylic acid, diisooctyl ester

20.89
1963448
5.56
620603
9.45
Heneicosane

20.97
441729
1.25
151709
2.31
Oxalic acid, 2-ethylhexyl pentadecyl ester

21.66
798268
2.26
78439
1.19
cis-13,16-Docasadienoic acid

22.25
84015
0.24
25211
0.38
(2,2,6-Trimethyl-bicyclo[4.1.0]hept-1-yl)-methanol

22.34
448534
1.27
81446
1.24
Tritetracontane

22.90
2376704
6.73
483543
7.36
Cyclopropanemethanol,. alpha.,2-dimethyl-2-(4-methyl-3-pentenyl)-, [1.alpha.(R),2.alpha.]-

23.70
925582
2.62
199546
3.04
Pentadecanoic acid

35337195
100.00
6570408
100.00

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%
Name

5.98
179688
0.89
14717
0.29
1-[t-Butyl]-3-[2-hydroxyethyl]-2-thiourea

6.53
206765
1.03
16890
0.33
1,3,3-Trimethylcyclohex-1-ene-4-carboxaldehyde, (+,−)-

6.99
477865
2.38
39661
0.77
Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-

7.75
17402
0.09
8436
0.16
2-Octenoic acid, 7-hydroxy-, ethyl ester

7.99
277810
1.38
25340
0.49
2-Cyclohexen-1-one, 3-methyl-

8.72
58849
0.29
11308
0.22
Decanoic acid, 3-methyl-

8.89
61164
0.30
11322
0.22
1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-, (Z)-

9.38
478340
2.38
65463
1.27
Borneol

9.84
13698
0.07
7413
0.14
1H-Cycloprop[e]azulene, decahydro-1,1,7-trimethyl-4-methylene-, [1aR-(1a.alpha.,4a.alpha.,7.alpha.,7a.beta.,7b. alpha.)]

9.88
11189
0.06
8044
0.16
Spiro[cyclopropane-1,8′(1H′)[3a.6]methanocyclopentacycloocten]-10′-one, octahydro-, (3'as,6'R,9'ar)

10.48
861378
4.28
116784
2.27
2-Butenoic acid, 2-methyl-, (E)-

10.99
54684
0.27
15680
0.31
Decane, 2-methyl-

11.29
23403
0.12
13933
0.27
Heptanoic acid

11.40
96098
0.48
29717
0.58
2-Hexanol, 3,3,5-trimethyl-2-(3-methylphenyl)-

11.62
96495
0.48
30081
0.59
n-Nonadecanol-1

12.34
43727
0.22
14425
0.28
Decane, 2-methyl-

12.58
72201
0.36
17495
0.34
E-6-Octadecen-1-ol acetate

12.96
95214
0.47
31350
0.61
1-Dodecanol

13.30
413748
2.06
114634
2.23
Tricyclo[3.1.0.0(2,4)]hexane, 3,6-diethyl-3,6-dimethyl-, trans-

13.56
58576
0.29
18742
0.37
2-Cyclohexene-1-methanol, 2,6,6-trimethyl-

13.81
333618
1.66
122979
2.40
Nerolidyl acetate

13.94
257018
1.28
76005
1.48
2,3-Dipropyl-cyclopropanecarboxylic acid, ethyl ester

14.00
27371
0.14
18033
0.35
Tetradecanoic acid, ethyl ester

14.15
101642
0.51
25064
0.49
10s,11s-Himachala-3(12),4-diene

14.24
103868
0.52
28501
0.56
2H-Pyran-2-one, 4-hydroxy-6-methyl-

14.36
65809
0.33
24124
0.47
Terpin Hydrate

14.66
219672
1.09
107246
2.09
2-Bromotetradecane

14.74
136624
0.68
55418
1.08
1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene-, [1ar-(1a.alpha.,4a.alpha.,7.beta.,7a.beta.,7b.alpha.)]

14.83
943433
4.69
399599
7.78
2-Pentadecanone, 6,10,14-trimethyl-

15.02
72288
0.36
29358
0.57
2-Piperidinone, 1-(3,4,5,6-tetrahydro-2-pyridinyl)-

15.11
221117
1.10
85066
1.66
7-Oxabicyclo[4.1.0]heptane, 2,2,6-trimethyl-1-(3-methyl-1,3-butadienyl)-5-methylene-

15.22
334667
1.66
83563
1.63
2H-Cyclopentacyclooctene, 4,5,6,7,8,9-hexahydro-1,2,2,3-tetramethyl-

15.34
21275
0.11
13398
0.26
Heptanoic acid, anhydride

15.82
179493
0.89
59327
1.16
1H-Cycloprop[e]azulene, decahydro-1,1,7-trimethyl-4-methylene-

16.04
2866546
14.25
855621
16.66
1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1.alpha.,4a.beta.,8a.alpha.)]-

16.41
159687
0.79
39983
0.78
9-Isopropyl-1-methyl-2-methylene-5-oxatricyclo[5.4.0.0(3,8)]undecane

16.59
474369
2.36
220498
4.29
Tricosane

16.78
432738
2.15
180735
3.52
Limonen-6-ol, pivalate

16.96
250742
1.25
76006
1.48
(−)-Spathulenol

17.12
144462
0.72
59742
1.16
Longipinocarveol, trans-

17.23
116967
0.58
29430
0.57
1-Cyclohexyl-1-(2-methylenecyclohexyl)ethanol

17.39
149266
0.74
44782
0.87
Cyclohexane, butyl-

17.57
118813
0.59
37221
0.72
(1-Ethylbuta-1,3-dienyl)benzene

17.91
52764
0.26
16286
0.32
Benzeneethanol,. alpha.-ethyl-

18.00
278732
1.39
107308
2.09
Dodecanoic acid

18.18
203495
1.01
68303
1.33
But-2-enoic acid, amide, 3-methyl-N-methallyl-

18.46
799323
3.97
240431
4.68
Heptacosane

18.65
168921
0.84
74823
1.46
9,12-Octadecadienoic acid, ethyl ester

19.46
224542
1.12
39309
0.77
Phytol

19.92
156623
0.78
28959
0.56
Acetic acid, 3-hydroxy-6-isopropenyl-4,8a-dimethyl-1,2,3,5,6,7,8,8a-octahydronaphthalen-2-yl ester

20.21
144167
0.72
40315
0.79
Oxacycloheptadec-8-en-2-one

20.31
219523
1.09
59841
1.17
Tetradecanoic acid

20.50
49486
0.25
17568
0.34
1,3-Dioxolane, 4,4,5-trimethyl-2-pentadecyl-

20.60
195477
0.97
54141
1.05
Fumaric acid, ethyl 2-methylallyl ester

20.80
253010
1.26
76165
1.48
Tetratetracontane

20.94
441843
2.20
129566
2.52
Oxalic acid, 2-ethylhexyl pentadecyl ester

21.05
123783
0.62
38025
0.74
Cyclohexane, 1,5-dimethyl-2,3-divinyl-

21.83
1095808
5.45
63024
1.23
1-Octacosanol

22.88
1803739
8.97
328164
6.39
Cyclohexan-1-ethanol, 1-hydroxymethyl-

23.70
2578313
12.82
469027
9.14
n-Hexadecanoic acid

20119328
100.00
5134389
100.00

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).

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).

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 CO₂. 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.

Conflicts of Interest

The author declares that they have no conflicts of interest.

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