Journal of Stress Physiology & Biochemistry, Vol. 18, No. 3, 2022, pp. 93-102 ISSN 1997-0838 Original Text Copyright © 2022 by Basel Saleh
OPEN ACCESS
ORIGINAL ARTICLE
GC/MS Artemisia herba alba Asso (Asteraceae) Phytochemical Screening
Basel Saleh
1 Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria.
*E-Mail: ascientific@aec. org.sy
Received April 12, 2022
Methanolic (Meth) and ethanolic (Eth) Artemisia herba-alba Asso aerial parts extract including buds (AB), leaves (AL) and flowers (AF) were phytochemicaly analyzed using gas chromatography-mass spectrometry (GC/MS) analysis. A. herba-alba GC/MS chromatogram showed 16 and 39 compounds were occurred in Meth and Eth A. herba-alba AB extract, respectively, revealing that the Thujone (37.026% and 49.022%), 9-Octadecanamide, (Z)-(15.471% and 11.479%) and Eucalyptol (10.057% and 10.083%) were presented as a major compounds for Meth and Eth A. herba-alba AB extract, respectively. Whereas, 24 and 20 compounds were occurred in Meth and Eth A. herba-alba AL extract, respectively; where, 9-Octadecanamide, (Z)- (28.687%), Phytol (12.611%) and Palmitoleamide (12.304%) were presented as a major compounds for Meth A. herba-alba AL extract. Whereas, they were 9-Octadecanamide, (Z)- (25.687%), Dodecanamide (16.142%) and Camphor (14.494%) presented as a major compounds for Eth A. herba-alba AL extract. As for AF, 28 and 14 compounds were occurred in Meth and Eth A. herba-alba AF extract, respectively; where, 9-Octadecanamide, (Z)- (25.623%), Eucalyptol (11.879%) and Hexadecanamide (10.771%) were presented as a major compounds for Meth A. herba-alba AF extract. Whereas, they were 9-Octadecanamide, (Z)- (23.295%), Hexadecanamide (16.452%) and Thujone (13.144%) presented as a major compounds for Eth A. herba-alba AF extract. The current study highlights different bioactive compounds make this species as a good candidate to be used as a cheap natural source in pharmacology and medicine applications. The current study highlights for the first time A. herba-alba phytochemical analysis in Syria.
Key words: Artemisia herba-alba, gas chromatography-mass spectrometry (GC/MS), Phytochemical analysis, 9-Octadecanamide, (Z)-, Hexadecanamide
Artemisia is a genus belongs to Asteraceae family, and includes approximately 300 species of small herbs and shrubs (Dob and Benabdelkader, 2006). In Syrian flora, Artemisia genus is represented with about 5 species, of which Artemisia herba-alba species wild grown in Syria (Mouterde 1983).
Artemisia herba-alba Asso, known as desert wormwood and as shTeh in Arabic. It is a perennial shrub commonly grows on the dry steppes of the Mediterranean regions in Northern Africa (Saharan Maghreb), Western Asia (Arabian Peninsula) and Southwestern Europe (USDA 2010).
Abou El-Hamd et al. (2010) reported that the sesquiterpene lactones, flavonoids, phenolic compounds & waxes and essential oils (EOs) were isolated and identified as the main secondary metabolites from A. herba-alba and other Artemisia species.
It has been reported that the Artemisia genus has an important role in folk medicine by many cultures
(Moufid and
More recently, Kshirsagar and Rao (2021) reviewed application of Artemisia sp. in medicine and pharmacology as antiviral and anti-inflammatory agents.
Of which, A. herba-alba herb exhibited many medicinal properties e.g. as antidiabetic,
insectidal,
antispasmodic, antihypertensive, antimalarial, anthelmintic, antileishmanial, nematicidal, neurological pesticidal, allelopathic and cytoprotective activities (Abou El-Hamd et al., 2010; Moufid and Eddouks, 2012; Janackovic et al., 2015; Riffi et al., 2020).
Moufid and Eddouks (2012) reported that A. herba alba biological activity could mainly related to its content of many bioactive compounds e.g. herbalbin, cis-chryanthenyl acetate, flavonoids (hispidulin and cirsilineol), monoterpenes and sesquiterpene.
Phytochemical screening of natural products presented in plants species is requested for any pharmaceutical and medicine researches and applications. In this regards, many different analytical
methods have been employed to determine Artemisia phytochemical constituents; e.g. fourier-transform infrared spectroscopy (FTIR) (Hameed et al., 2016); high-performance liquid chromatography (HPLC) (Bourgou et al., 2015); ultra-performance liquid chromatography (UPLC) coupled to photodiode array detection (PDA) and mass spectrometry (MS) (UPLC-PDA-MS) (Dane et al. 2016); gas chromatography-mass spectrometry (GC/MS) (Vernin et al., 1995; Bourgou et al., 2015; Janackovic et al., 2015; Parameswari and Devika, 2017; Nasser and Arnold-Apostolides, 2018; Riffi et al., 2020) and liquid chromatography (LC) coupled to mass spectrometry (MS) (LC/MS) (Mamatova et al., 2019).
Little is known about phytochemical screening of A. herba-alba species in Syria. Thereby, the presented study focused on its phytochemical analysis during different development stages using GC/MS analysis for the first time.
MATERIAL AND METHODS
Plant materials
Buds (AB), leaves (AL) and flowers (AF) Artemisia herba-alba Asso aerial parts (10 plants/sample) were collected separately from wild A. herba-alba species grown in its natural habitat from rural Damascus regions-Syria (altitude of 950 m and annual rainfall of 240 mm). Samples were shade dried for two weeks, powdered by special electric mill and stored separately in glass bowls until extracts preparation.
Extracts preparation
The fine powder for each sample was extracted with methanol and ethanol solvents, separately as flowing: 1 g of fine powder was extracted with 10 mL solvent overnight, filtrated with filter papers (Whatman no.1). Then, all extracts were kept in tightly fitting stopper bottles and stored at 4 °C. The final obtained extracts were then analyzed using GC/MS analysis.
GC/MS analysis
GC Chromatec-Crystal 5000 system, supported with Chromatec Crystal Mass Spectrometry Detector (Chromatec, Russia) has been employed to investigate phytochemical methanolic and ethanolic A. herba-alba
since ancient times (European medicine, North Africa and Arabic traditional medicine)
Eddouks, 2012).
antimicrobial, antioxidant, antiradical,
aerial parts extracts analysis. GC/MS analysis has been performed according to the following conditions: The range scan was 42-850 MU, the column [(BP-5-MS (30 m x 0.25 mm x 0.25 |jm)], carrier gas (0.695 ml/min flow of Helium gas). Oven temperature was programmed initially at 35 °C for 1 min, then an increase by 10°C /1 min till 220 °C, then increase to 230 °C by 1°C /1 min followed by 10 °C /1 min increasing till 255 °C (hold for 5 min). Injector temperature was 275 °C and detector temperature was 280 °C and ionization energy was 70 ev. Each extract component was identified by comparing retention time values of gas chromatography on polar columns and by comparing mass spectrum and NIST-17 library databases.
RESULTS AND DISCUSSION
A. herba-alba GC/MS analysis revealed 16 and 39 compounds were occurred in methanolic and ethanolic A. herba-alba buds extract, respectively; of which, 8 compounds were common for the two both buds extract. It has been found that the Thujone (37.026% and 49.022%), 9-Octadecanamide, (Z)- (15.471% and 11.479%) and Eucalyptol (10.057% and 10.083%) were presented as a major compounds for methanolic and ethanolic A. herba-alba buds extract, respectively (Tables 1 & 2).
Whereas, 24 and 20 compounds were occurred in methanolic and ethanolic A. herba-alba leaves extract, respectively; of which, 9 compounds were common for the two both leaves extract. It has been found that the 9-Octadecanamide, (Z)- (28.687%), Phytol (12.611%) and Palmitoleamide (12.304%) were presented as a major compounds for methanolic A. herba-alba leaves extract (Table 3). Whereas, they were 9-Octadecanamide, (Z)-(25.687%), Dodecanamide (16.142%) and Camphor (14.494%) presented as a major compounds for ethanolic A. herba-alba leaves extract (Table 4).
As for flowers parts, 28 and 14 compounds were occurred in methanolic and ethanolic A. herba-alba flowers extract, respectively; of which, 9 compounds were common for the two flowers extract. It has been found that the 9-Octadecanamide, (Z)- (25.623%), Eucalyptol (11.879%) and Hexadecanamide (10.771%)
were presented as a major compounds for methanolic A. herba-alba flowers extract (Table 5). Whereas, they were 9-Octadecanamide, (Z)- (23.295%), Hexadecanamide (16.452%) and Thujone (13.144%) presented as a major compounds for ethanolic A. herba-alba flowers extract (Table 6).
Extracts of wild A. herba-alba aerial parts (buds AB, leaves AL and flowers AF) grown in rural Damascus regions-Syria, were phytochemically analyzed using GC/MS technique.
It worth noting that the 9-Octadecatrienoic acid (Z), tetradecyl ester and Agaricic acid compounds presented in ethanolic A. herba-alba buds extract in the current study were presented in methanolic A. nilagirica leaves extract using GC/MS analysis (Parameswari and Devika 2017).
Vernin et al. (1995) reported that the camphor (1948%), 1,8-cineole (5-20%), chrysanthenone (5-22.5%), a-thujone (1.0-26.7%), p-thujone (1.65-9.3%) and camphene (1.7-7.9%) were mainly presented in Algerian A. herba alba EOs using GC/MS analysis. Whereas, Zouari et al. (2010) reported that cis-chrysantenyl acetate (10.60%), sabinyl acetate (9.13%) and a-thujone (8.73%) were the major compounds presented in leaves and flowers Tunisian A. herba-alba EOs. Moreover, Abou-Darwish et al. (2015) reported that p-Thujones (25.1%), a-Thujones (22.9%), Eucalyptol (20.1%) and Camphre (10%) were the major compounds presented in Jordanian A. herba-alba EOs. Indeed, El-Seedi et al. (2017) reported that Piperitone (26.5%), ethyl cinnamate (9.5%), camphor (7.7%) and hexadecanoic acid (6.9%) were the major compounds recorded in Egyptian A. herba-Alba leaves EOs. Similarly, Bourgou et al. (2015) reported that Camphor (0.64- 31.51 %), a-Thujone (11.62- 13.93%), Fenchol (7.51- 13.85%) and Nordavanone (1.26-9.44%) were the major compounds presented in Tunisian A. herba-Alba EOs using GC/MS analysis. Whereas, p-Coumaric acid (6.19-23.34%), Naringenin (3.36-20.19%) and Caffeic acid (1.32-14.04%) were presented in methanolic A. herba-Alba extract using HPLC analysis.
Janackovic et al. (2015) reported that the Camphor (24.7%), Chamazulene (20.9%), Isomer C14H18 (6.3%) and Bornyl acetate (4.9%) were mainly presented in A.
arborescens EOs. Whereas, Chrysanthenone (20.5%) and cis-Chrysanthenyl acetate (17.7%) were mainly presented in A. herba-alba EOs. While, Piperitone (30.2%), cis-Chrysanthenol (9.1%) and Davana ether (7.9%) were mainly presented in A. judaica EOs using GC/MS analysis. While, Parameswari and Devika (2017) reported that Ergosta-5, 7, 22-trien- 3- o1, acetate, (3a, 22E), Agaricic acid, Bufa- 20, 22-dienolide, 3, 14-dihydroxy- (3a, 5a) and 9-Octadecenoic acid (Z)-tetradecyl ester, were the majors constituents presented in methanolic A. nilagirica leaves extract using GC/MS analysis. Whereas, Mamatova et al. (2019) reported the occurrence of flavonoids: apigenin, luteolin, rutin, two O-methylated flavonols (isorhamnetin & rhamnazine), coumarin compounds (umbelliferone, scopoletin and scopolin (scopoletin 7-glucoside), 3-hydroxycoumarin and 4-hydroxycoumarin), chlorogenic acid and two dicaffeoylquinic acid isomers in ethanolic and chloroform A. gmelinii extracts using LC/MS analysis. Moreover, Siddiqui et al. (2018) reported the occurrence of alkaloids, flavonoids, saponin, tannins, steroids, glycosides and phenols in the twelve different solvents extract of A. annua.
Nasser and Arnold-Apostolides (2018) reported that the a-pinene (45.89%), borneol (11.3%) and 1,8-cineole (10.8%) were the most abundant compounds in the A. herba-alba EOs; whereas, camphene (15.71%), myrtenal (6.47%) and m-cymene (5.97%) were the most abundant compounds in its ethanolic extract; while camphor (32.91%), 1,8-cineole (9.98%) and borneol (6.78%) were the most abundant compounds in its acetonic extract, using GC/MS analysis. Recently, Riffi et al. (2020) reported that the Camphor (96.15%), Caryophyllene oxide (29,45%), Santoline alcohol (22.56%), 10,12-Octadecadienoic acid (20.68%) and Chrysanthyl acetate (16.82%) were the major compounds presented in the 4 fractions (F1, F2, F3 and F4) of A. herba alba EOs using GC/MS analysis.
It worth noting that in the current study, Eucalyptol content ranged between 5.392-11.879%; whereas, this
compound was recorded to be 20.1% in Jordanian A. herba-alba EOs (Abou-Darwish et al., 2015). Otherwise, Camphor content ranged between 0.315-14.494% in the current study, whereas, this compound was ranged between 19-48% in Algerian A. herba alba EOs (Vernin et al., 1995); 7.7% in Egyptian A. herba-Alba leaves EOs (El-Seedi et al., 2017); between 0.64- 31.51 % in Tunisian A. herba-Alba EOs (Bourgou et al., 2015); 24.7, 1.8 and 0.3% in Libyan A. arborescens, A. herba-alba and A. judaica EOs, respectively (Janackovic et al., 2015); 32.91% in Lebanon acetonic A. herba-alba extract (Nasser and Arnold-Apostolides, 2018) and 96.15% in Moroccan A. herba-Alba EOs (Riffi et al., 2020). Moreover, Camphene content in the current study was ranged between 0.184-1.028%, whereas, this compound was ranged between 1.7-7.9% in Algerian A. herba alba EOs (Vernin et al., 1995); 1.6, 0.7 and 0% in Libyan A. arborescens, A. herba-alba and A. judaica EOs, respectively (Janackovic et al., 2015) and 15.71% in Lebanon ethanolic A. herba-alba extract (Nasser and Arnold-Apostolides, 2018). Indeed, p-Cymene in the current study was recorded to be 0.422%, whereas it was recorded to be 0.5, 0.5 and 1.7% in Libyan A. arborescens, A. herba-alba and A. judaica EOs, respectively (Janackovic et al., 2015) and 5.97% in Lebanon ethanolic A. herba-alba extract (Nasser and Arnold-Apostolides, 2018). Moreover, Caryophyllene oxide in the current study was ranged between 1.1813.639%, whereas, it was recorded to be 0.2 % in Libyan A. arborescens EOs along with its absence in Libyan A. herba-alba and A. judaica EOs (Janackovic et al., 2015) and 29,45% in Moroccan A. herba-Alba EOs (Riffi et al., 2020).
These differences in compounds content could be attributed to many factors like e.g. studied Artemisia species and substrate type, where in the current study, extracts have been prepared with solvents whereas, for the other studies they were EOs. Moreover, as known geographical distribution play an important role as a main factor affecting phytochemical composition (Zhang et al, 2017).
Table 1: GC/MS spectrum of methanolic A. herba-alba Asso buds extract.
Peak No RT (min) Name of Compound Peak area (%)
1 9.505 Eucalyptol 10.057
2 10.695 Thujone 37.026
3 10.865 Bicyclo[3.1.0]hexan-3-one,4-methyl-1-(1-methylethyl)- 4.631
4 11.344 p-Mentha-1,8-dien-7-ol 1.876
5 17.851 Jasmonic acid 0.348
6 21.400 n-Hexadecanoic acid 0.821
7 23.558 Hexadecanamide 1.255
8 24.252 Palmitoleamide 4.402
9 25.074 Octadecanamide 8.527
10 26.776 Caryophyllene oxide 1.868
11 29.513 9-Octadecanamide, (Z)- 15.471
12 30.159 Octadecanamide 3.324
13 30.446 ß-Guaiene 2.155
14 31.987 1-Heptatriacotanol 0.991
15 32.826 Corymbolone 6.500
16 33.773 13-Docosenamide, (Z)- 0.746
Table 2: GC/MS spectrum of ethanolic A. herba-alba Asso buds extract.
Peak No RT (min) Name of Compound Peak area (%)
1 6.068 Ethylene glycol diglycidyl ether 0.180
2 7.257 a-Pinene 0.989
3 7.845 trans-ß-Ocimene 0.077
4 8.127 Camphene 0.184
5 8.458 3-Carene 0.211
6 9.263 p-Cymene 0.422
7 9.501 Eucalyptol 10.083
8 10.012 R-Limonene 0.144
9 10.101 p-Menth-8-en-1-ol, steroisomer 0.125
10 10.704 Thujone 49.022
11 11.229 Camphor 0.558
12 11.342 p-Mentha-1,8-dien-7-ol 2.708
13 12.109 4-Hydoxy-a-thujone 0.419
14 12.579 Methyl 10,11-tetradecadienoate 0.133
15 12.939 cis-p-Mentha-2,8-dien-1-ol 0.689
16 14.271 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 0.194
17 14.504 9,12-Octadecadienoyl chloride, (Z,Z)- 0.166
18 15.327 Caryophyllane,4,8-ß-epoxy 0.397
19 24.700 Hexadecanamide 0.691
20 25.047 Octadecanamide 5.929
21 26.745 Picrotoxinin 1.431
22 27.474 Olean-12-ene-3,28-diol, (3ß)- 0.205
23 28.804 9-Octadecatrienoic acid (Z), tetradecyl ester 0.151
24 28.976 cis-11-Eicosenamide 0.186
98 GC/MS Artemisia herba alba Asso Phytochemical Screening...
25 29.480 9-Octadecanamide, (Z)- 11.283
26 30.138 Hexadecanamide 2.061
27 30.404 Xanthumin 1.837
28 30.987 9-Hexadecanoic acid, eicosyl ester, (Z)- 0.120
29 31.445 Ergosta-5,22-dien-3-ol,acetate, (3B,22E)- 0.131
30 31.956 1-Heptatriacotanol 1.031
31 32.250 9-Octadecanenitrile, (Z)- 0.196
32 32.388 B-Santanol acetate 0.276
33 32.786 Corymbolone 6.188
34 32.986 Ethyl iso-allocholate 0.114
35 33.762 Agaricic acid 0.479
36 33.902 cis-9,10-Epoxyoctadecanamide 0.213
37 34.314 Deoxyspergualin 0.161
38 34.756 Triaziquone 0.378
39 34.958 7-Heptadecene, 17-chloro- 0.239
Table 3: GC/MS spectrum of methanolic A. herba-alba Asso leaves extract.
Peak No RT (min) Name of Compound Peak area (%)
1 8.130 Camphene 0.427
2 9.505 Eucalyptol 5.392
3 10.685 Bicyclo 0.645
4 10.854 Bicyclo[3.1.0]hexan-3-one,4-methyl-1-(1-methylethyl)- 1.417
5 11.356 p-Mentha-1,8-dien-7-ol 4.660
6 11.753 Camphor 0.315
7 12.941 Isoborneol 1.168
8 17.843 (+)-cis-Verbenol, acetate 2.281
9 21.404 Butanoic acid, octyl ester 1.905
10 23.533 n-Hexadecanoic acid 0.326
11 23.784 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- 0.307
12 24.269 Phytol 12.611
13 25.063 Palmitoleamide 12.304
14 25.549 Dodecanamide 1.274
15 26.799 Corymbolone 6.218
16 27.483 B-Neoclovene 1.157
17 28.037 13-Docosenamide, (Z)- 2.137
18 29.530 9-Octadecanamide, (Z)- 28.687
19 29.980 Caryophyllene oxide 1.181
20 30.163 Hexadecanamide 4.504
21 30.435 B-Guaiene 4.462
22 32.756 1-Heptatriacotanol 4.659
23 33.773 13-Docosenamide, (Z)- 1.356
24 33.905 Palmitoleamide 0.607
Table 4: GC/MS spectrum of ethanolic A. herba-alba Asso leaves extract.
Peak No RT (min) Name of Compound Peak area (%)
1 6.063 3-Nitropropanoic acid 1.270
2 8.159 Camphene 1.028
3 9.493 Eucalyptol 8.789
4 10.681 Thujone 2.581
5 10.858 Bicyclo[3.1.0]hexan-3-one,4-methyl-1-(1-methylethyl)- 0.459
6 11.348 Camphor 14.494
7 11.745 Isoborneol 0.710
8 12.937 Carveol 2.176
9 13.359 Isobornyl acetate 0.484
10 20.628 Hexadecanenitrile 0.739
11 20.894 2(3H)-Furanone, 5-dodecyldihydro- 0.750
12 22.278 Pentadecanal- 1.427
13 25.003 Dodecanamide 16.142
14 26.713 1-Heptatriacotanol 7.920
15 27.479 Nootkatone 1.428
16 29.434 9-Octadecanamide, (Z)- 25.687
17 29.933 Urs-12-ene 1.397
18 30.117 Palmitoleamide 3.541
19 30.368 Xanthumin 5.325
20 32.687 9,12,15-Octadecatrienoic acid, 2,3-dihyroxypropyl ester, (Z,Z,Z)- 3.651
Table 5: GC/MS spectrum of methanolic A. herba-alba Asso flowers extract .
Peak No RT (min) Name of Compound Peak area (%)
1 8.125 Camphene 0.323
2 9.506 Eucalyptol 11.879
3 10.691 Thujone 4.000
4 10.867 Bicyclo[3.1.0]hexan-3-one,4-methyl-1-(1-methylethyl)- 3.139
5 11.157 Bornyl chloride 0.362
6 11.356 Camphor 2.815
7 11.479 7-0xabicyclo[4.1.0]heptane, 1-methyl-4-(2-methyloxiranyl)- 0.398
8 11.680 3-Buten-2-one, 4-(3-cyclohexane-1-yl)- 0.461
9 11.756 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- 0.482
10 12.115 4-Hydroxy-ß-Thujone 0.403
11 12.950 p-Mentha-1(7),8(10)-dien-7-ol 0.537
12 21.423 n-Hexadecanoic acid 4.232
13 23.533 9-Octadecanoic acid (Z)-, methyl ester 0.197
14 23.775 Dodecanamide 1.366
15 24.281 17-Octadecynoic acid 9.805
16 25.070 Hexadecanamide 10.771
17 25.256 Picrotoxin 0.631
18 26.791 Caryophyllene oxide 3.366
19 27.495 Aromandendrene 0.513
20 27.729 Palmitoleamide 2.649
21 29.538 9-Octadecenamide, (Z)- 25.623
22 30.157 Octadecenamide 3.661
23 30.429 ß-Guaiene 1.280
24 31.452 1-Heptatriacotanol 1.196
25 31.981 Cholestan-3-ol, 2-methylene-, (3ß,5a)- 0.772
26 32.784 Corymbolone 7.145
27 33.766 9-Hexadecanoic acid 1.456
28 33.917 13-Docosenamide, (Z)- 0.538
Table 6: GC/MS spectrum of ethanolic A. herba-alba Asso flowers extract.
Peak No RT (min) Name of Compound Peak area (%)
1 9.493 Eucalyptol 9.408
2 10.687 Thujone 13.144
3 10.861 Bicyclo[3.1.0]hexan-3-one,4-methyl-1-(1-methylethyl)- 9.479
4 11.358 Camphor 6.729
5 20.628 Hexadecanenitrile 0.608
6 24.998 Hexadecanamide 16.452
7 26.701 Caryophyllene oxide 3.639
8 29.419 9-Octadecenamide, (Z)- 23.295
9 30.088 Palmitoleamide 5.560
10 30.360 Ethyl iso-allocholate 1.473
11 31.916 Cucurbitacin b, 25-desacetoxy- 1.730
12 32.234 Oleic acid 1.123
13 32.677 Corymbolone 6.462
14 33.735 Deoxyspergualin 0.898
CONCLUSION
GC/MS A. herba-alba aerial parts extracts chromatogram revealed that the 9-Octadecanamide, (Z)-was presented as a common and major compound in all studied parts extracts regardless tested solvent. The different bioactive compounds mainly occurred in A. herba-alba aerial parts extracts like 9-Octadecanamide, (Z)-, Thujone, Eucalyptol, Palmitoleamide, Hexadecanamide and others, make them as potential natural sources to be used in different pharmacology and medicine applications with low cost.
ACKNOWLEDGEM ENTS
I thank Dr. I. Othman (Director General of AECS) and Dr. N. Mirali (Head of Molecular Biology and Biotechnology Department in AECS) for their support, and also the Plant Biotechnology group for technical assistance.
CONFLICTS OF INTEREST
The author declare that they have no potential conflicts of interest.
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