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

Authors: Layth Sareea Al-Rekaby
Journal: Journal of Botany
 (2018)

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## 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.


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## Body

## 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) (Table1) 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
10–50µm
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/cmTable 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].


## 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 Table3 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 ofHibiscus 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
7.97The 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 (Table4). 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
100.00Figure 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 (Table5). 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
100.00Figure 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 Table6 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
100.00Figure 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 (Table7). 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
100.00Figure 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 (Table8). 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
1
7.02
765601
2.17
56572
0.86
Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-
2
8.74
82130
0.23
15029
0.23
Pentanoic acid, 4-methyl-
3
8.87
81976
0.23
10541
0.16
1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-
4
9.38
520061
1.47
49940
0.76
Isoborneol
5
9.87
32654
0.09
10191
0.16
Caryophyllene
6
9.91
9809
0.03
5883
0.09
Geranyl ethyl ether 2
7
10.44
1145521
3.24
132334
2.01
2-Butenoic acid, 2-methyl-, (E)-
8
11.01
145689
0.41
24839
0.38
Dodecane, 2,6,11-trimethyl-
9
11.29
78677
0.22
17466
0.27
Hexanoic acid
10
11.66
64866
0.18
19188
0.29
1-Heneicosanol
11
12.35
39374
0.11
14212
0.22
Heptadecane, 2,6,10,15-tetramethyl-
12
12.69
106924
0.30
11102
0.17
2-Butanone, 4-(2,2,6-trimethylcyclohexyl)-
13
13.30
356944
1.01
73595
1.12
Caryophyllene oxide
14
13.56
91558
0.26
33046
0.50
Octadecane, 1-chloro-
15
13.82
291034
0.82
89776
1.37
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-
16
14.66
168921
0.48
78432
1.19
Heneicosane
17
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.)]
18
14.83
408734
1.16
176706
2.69
2-Pentadecanone, 6,10,14-trimethyl-
19
15.09
467380
1.32
68319
1.04
1-Heneicosanol
20
15.26
319379
0.90
83584
1.27
Caprolactam
21
15.67
32473
0.09
18890
0.29
2-Bromotetradecane
22
16.05
318485
0.90
143648
2.19
Selina-6-en-4-ol
23
16.09
300889
0.85
133271
2.03
Hexadecanoic acid, ethyl ester
24
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.)]
25
16.60
473246
1.34
223525
3.40
Heneicosane
26
16.79
283275
0.80
117213
1.78
8-Hydroxy-2,2,8-trimethyldeca-5,9-dien-3-one
27
17.49
92528
0.26
39301
0.60
Hexadecane, 2,6,11,15-tetramethyl-
28
18.00
293141
0.83
105445
1.60
Eicosanoic acid
29
18.19
136422
0.39
55398
0.84
2-Decene, 2,4-dimethyl-
30
18.31
447480
1.27
149416
2.27
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-
31
18.51
2937254
8.31
1043306
15.88
Eicosane, 10-methyl-
32
18.80
151566
0.43
54379
0.83
2,6-Octadienal, 2,6-dimethyl-8-(tetrahydro-2H-2-pyranyloxy)
33
19.57
5322093
15.06
604327
9.20
Hentriacontane
34
20.75
11971493
33.88
1173046
17.85
1,2-Benzenedicarboxylic acid, diisooctyl ester
35
20.89
1963448
5.56
620603
9.45
Heneicosane
36
20.97
441729
1.25
151709
2.31
Oxalic acid, 2-ethylhexyl pentadecyl ester
37
21.66
798268
2.26
78439
1.19
cis-13,16-Docasadienoic acid
38
22.25
84015
0.24
25211
0.38
(2,2,6-Trimethyl-bicyclo[4.1.0]hept-1-yl)-methanol
39
22.34
448534
1.27
81446
1.24
Tritetracontane
40
22.90
2376704
6.73
483543
7.36
Cyclopropanemethanol,. alpha.,2-dimethyl-2-(4-methyl-3-pentenyl)-, [1.alpha.(R∗),2.alpha.]-
41
23.70
925582
2.62
199546
3.04
Pentadecanoic acid
35337195
100.00
6570408
100.00Table 9
Bioactive constituents of water extract of calyxes ofHibiscus 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
1
5.98
179688
0.89
14717
0.29
1-[t-Butyl]-3-[2-hydroxyethyl]-2-thiourea
2
6.53
206765
1.03
16890
0.33
1,3,3-Trimethylcyclohex-1-ene-4-carboxaldehyde, (+,−)-
3
6.99
477865
2.38
39661
0.77
Bicyclo[2.2.1]heptan-2-one, 1,7,7-trimethyl-, (1S)-
4
7.75
17402
0.09
8436
0.16
2-Octenoic acid, 7-hydroxy-, ethyl ester
5
7.99
277810
1.38
25340
0.49
2-Cyclohexen-1-one, 3-methyl-
6
8.72
58849
0.29
11308
0.22
Decanoic acid, 3-methyl-
7
8.89
61164
0.30
11322
0.22
1,6,10-Dodecatriene, 7,11-dimethyl-3-methylene-, (Z)-
8
9.38
478340
2.38
65463
1.27
Borneol
9
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.)]
10
9.88
11189
0.06
8044
0.16
Spiro[cyclopropane-1,8′(1H′)[3a.6]methano[3ah]cyclopentacycloocten]-10′-one, octahydro-, (3'as,6'R,9'ar)
11
10.48
861378
4.28
116784
2.27
2-Butenoic acid, 2-methyl-, (E)-
12
10.99
54684
0.27
15680
0.31
Decane, 2-methyl-
13
11.29
23403
0.12
13933
0.27
Heptanoic acid
14
11.40
96098
0.48
29717
0.58
2-Hexanol, 3,3,5-trimethyl-2-(3-methylphenyl)-
15
11.62
96495
0.48
30081
0.59
n-Nonadecanol-1
16
12.34
43727
0.22
14425
0.28
Decane, 2-methyl-
17
12.58
72201
0.36
17495
0.34
E-6-Octadecen-1-ol acetate
18
12.96
95214
0.47
31350
0.61
1-Dodecanol
19
13.30
413748
2.06
114634
2.23
Tricyclo[3.1.0.0(2,4)]hexane, 3,6-diethyl-3,6-dimethyl-, trans-
20
13.56
58576
0.29
18742
0.37
2-Cyclohexene-1-methanol, 2,6,6-trimethyl-
21
13.81
333618
1.66
122979
2.40
Nerolidyl acetate
22
13.94
257018
1.28
76005
1.48
2,3-Dipropyl-cyclopropanecarboxylic acid, ethyl ester
23
14.00
27371
0.14
18033
0.35
Tetradecanoic acid, ethyl ester
24
14.15
101642
0.51
25064
0.49
10s,11s-Himachala-3(12),4-diene
25
14.24
103868
0.52
28501
0.56
2H-Pyran-2-one, 4-hydroxy-6-methyl-
26
14.36
65809
0.33
24124
0.47
Terpin Hydrate
27
14.66
219672
1.09
107246
2.09
2-Bromotetradecane
28
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.)]
29
14.83
943433
4.69
399599
7.78
2-Pentadecanone, 6,10,14-trimethyl-
30
15.02
72288
0.36
29358
0.57
2-Piperidinone, 1-(3,4,5,6-tetrahydro-2-pyridinyl)-
31
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-
32
15.22
334667
1.66
83563
1.63
2H-Cyclopentacyclooctene, 4,5,6,7,8,9-hexahydro-1,2,2,3-tetramethyl-
33
15.34
21275
0.11
13398
0.26
Heptanoic acid, anhydride
34
15.82
179493
0.89
59327
1.16
1H-Cycloprop[e]azulene, decahydro-1,1,7-trimethyl-4-methylene-
35
16.04
2866546
14.25
855621
16.66
1-Naphthalenol, decahydro-1,4a-dimethyl-7-(1-methylethylidene)-, [1R-(1.alpha.,4a.beta.,8a.alpha.)]-
36
16.41
159687
0.79
39983
0.78
9-Isopropyl-1-methyl-2-methylene-5-oxatricyclo[5.4.0.0(3,8)]undecane
37
16.59
474369
2.36
220498
4.29
Tricosane
38
16.78
432738
2.15
180735
3.52
Limonen-6-ol, pivalate
39
16.96
250742
1.25
76006
1.48
(−)-Spathulenol
40
17.12
144462
0.72
59742
1.16
Longipinocarveol, trans-
41
17.23
116967
0.58
29430
0.57
1-Cyclohexyl-1-(2-methylenecyclohexyl)ethanol
42
17.39
149266
0.74
44782
0.87
Cyclohexane, butyl-
43
17.57
118813
0.59
37221
0.72
(1-Ethylbuta-1,3-dienyl)benzene
44
17.91
52764
0.26
16286
0.32
Benzeneethanol,. alpha.-ethyl-
45
18.00
278732
1.39
107308
2.09
Dodecanoic acid
46
18.18
203495
1.01
68303
1.33
But-2-enoic acid, amide, 3-methyl-N-methallyl-
47
18.46
799323
3.97
240431
4.68
Heptacosane
48
18.65
168921
0.84
74823
1.46
9,12-Octadecadienoic acid, ethyl ester
49
19.46
224542
1.12
39309
0.77
Phytol
50
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
51
20.21
144167
0.72
40315
0.79
Oxacycloheptadec-8-en-2-one
52
20.31
219523
1.09
59841
1.17
Tetradecanoic acid
53
20.50
49486
0.25
17568
0.34
1,3-Dioxolane, 4,4,5-trimethyl-2-pentadecyl-
54
20.60
195477
0.97
54141
1.05
Fumaric acid, ethyl 2-methylallyl ester
55
20.80
253010
1.26
76165
1.48
Tetratetracontane
56
20.94
441843
2.20
129566
2.52
Oxalic acid, 2-ethylhexyl pentadecyl ester
57
21.05
123783
0.62
38025
0.74
Cyclohexane, 1,5-dimethyl-2,3-divinyl-
58
21.83
1095808
5.45
63024
1.23
1-Octacosanol
59
22.88
1803739
8.97
328164
6.39
Cyclohexan-1-ethanol, 1-hydroxymethyl-
60
23.70
2578313
12.82
469027
9.14
n-Hexadecanoic acid
20119328
100.00
5134389
100.00Figure 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 ofHibiscus 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 (Table1) 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 (Table2). 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.



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*Source: 9097363-2018-02-11.xml*