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Journal of Stress Physiology & Biochemistry, Vol. 19, No. 1, 2023, pp. 97-104 ISSN 1997-0838 Original Text Copyright © 2022 by Basel Saleh
ORIGINAL ARTICLE
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Phytochemical Analysis of Two Achillea (Asteraceae) Species Using GC/MS Technique
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 September 29, 2022
Wild Achillea aleppica DC and Achillea arabica Kotschy flowering, aromatic and perennial herbs, grown in the Middle-Southern regions of Syria were assessed for their ethanolic and acetonic aerial parts extracts phytochemical analysis based on gas chromatography-mass spectrometry (GC/MS) analysis. Overall, GC/MS chromatogram revealed that the 9-Octadecenamide, (Z)- (41.656% and 61.097%) and Hexadecanamide (36.542% and 20.238%) were the most abundant compounds for ethanolic and acetonic A. aleppica aerial parts extracts, respectively. Whereas, 9-Octadecenamide, (Z)- (41.280% and 53.990%) and Hexadecanamide (30.828% and 14.445%) were the most abundant compounds for ethanolic and acetonic A. Arabica aerial parts extracts, respectively. This study could consider as the first report highlights A. aleppica and A. arabica extracts phytochemical analysis.
Key words: Achillea aleppica; Achillea arabica (Achillea biebersteinii); Gas chromato-graphy-mass spectrometry (GC/MS); Phytochemical analysis; 9-Octadecenamide,
(Z)-
aqueous ethanolic (40 % v/v) vegetative parts A.
Achillea genus belongs to Asteraceae family the largest angiosperms's family, comprise approximately 1500 genera and 23000 species. They spilled in three subfamilies and seventeen tribes. This genus involved 115 species of perennial herbs; all of them are native to temperate regions of the northern hemisphere (Moradkhani et al. 2012).
Achillea species exhibited wide range in medicine and pharmaceutical applications; e.g. as antimicrobial (Stojanovic et al. 2005; Toncer et al. 2010; Tabanca et al. 2011; Albayrak and Silahtarlioglu 2019); antioxidant (Toncer et al. 2010; Manayi et al. 2012; Polatoglu et al. 2013; Albayrak and Silahtarlioglu 2019); insecticidal (Toncer et al. 2010; Tabanca et al. 2011; Polatoglu et al. 2013); herbicidal (Toncer et al. 2010; Polatoglu et al. 2013); cytotoxic (Albayrak and Silahtarlioglu 2019); antinociceptive and anti-inflammatory (Toncer et al. 2010) properties. Moreover, they used in traditional remedies against rheumatic pain and digestive complaints, fever, common cold, pneumonia and hemorrhage (Manayi et al. 2012).
The genus Achillea is represented in Syrian Flora with about 9 species (Mouterde 1983), of which A. aleppica DC and A. arabica Kotschy (Synonyms. Achillea biebersteinii Afanasiev) were wild grown in Syria. A. aleppica DC has antimicrobial, anti-inflammatory and antinociceptive properties (Toncer et al. 2010; Tabanca et al. 2011). Whereas, A. arabica Kotschy has hepatoprotective, antioxidant, herbicidal and insecticidal properties (Toncer et al. 2010; Tabanca et al. 2011; Ba§er 2016; Al-Said et al. 2016).
Different analytical methods allowed for long time identifying chemical compounds occurred in plants essential oils (EOs) and extracts. Of which, GC/MS analysis has been extensively employed worldwide for this purpose; e.g. in A. millefolium, A. lingulata, A. holosericea and A. clavennae species EOs (Boskovic et al. 2005); A. biebersteinii, A. millefolium and A. wilhelmsii EOs (Dehghan and Elmi 2014); A. biebersteinii EOs (Al-Said et al. 2016; Sevindiki et al. 2018); A. fragrantissima EOs (Hatem et al. 2018); A. coarctata EOs (Albayrak and Silahtarlioglu 2019) and
micrantha extract (Astafyeva et al. 2018).
To our knowledge, the majority of researches on A. aleppica DC and A. arabica Kotschy species phytochemical analysis have been focused on their essential oils composition. However, little is known about their extracts phytochemical analysis. Thereby, the current investigation focused on their ethanolic and acetonic aerial parts extracts phytochemical analysis using GC/MS analysis.
MATERIALS AND METHODS
Plant materials and preparation of extracts
Aerial parts of A. aleppica and A. arabica (10 plants for each species) were collected and bulked as representative for each Achillea sp. Samples have been collected during blooming stage from two wild Achillea species grown in their natural habitat from Middle-Southern regions in Syria. Achillea aleppica DC was collected from rural Damascus regions - Syria; whereas, Achillea arabica Kotschy was collected from rural Homs regions - Syria (Table 1).
Samples were shade dried for two weeks, and were milled to fine powder by special electric mill and stored separately in glass bowls for ethanolic and acetonic extracts preparation.
The fine powder for each sample was extracted with ethanol and acetone 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 subjected to 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 ethanolic and acetonic A. aleppica and A. arabica 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 Mm)], carrier gas (0.695 ml/min flow of Helium gas). Oven temperature
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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
In the current study, GC/MS chromatogram revealed 11 and 23 compounds in ethanolic and acetonic A. aleppica aerial parts extracts, respectively (Tables 2 & 3). Of which, 9-Octadecenamide, (Z)- (41.656%), Hexadecanamide (36.542%) and Tetradecanamide (9.965%) were the most abundant compounds in ethanolic A. aleppica aerial parts extracts (Table 2). Whereas, 9-Octadecenamide, (Z)- (61.097%), Hexadecanamide (20.238%) and Nonadecanamide (5.601%) were the most abundant compounds in acetonic A. aleppica aerial parts extracts (Table 3). As for A. Arabica, they were 8 and 34 compounds in ethanolic and acetonic A. aleppica aerial parts extracts, respectively (Tables 4 & 5). Of which, 9-Octadecenamide, (Z)- (41.280%), Hexadecanamide (30.828%) and Dodecanamide (8.940%) were the most abundant compounds in ethanolic A. Arabica aerial parts extracts (Table 4). Whereas, 9-Octadecenamide, (Z)-(53.990%), Hexadecanamide (14.445%) and 13-Docosenamide, (Z)- (7.829%) were the most abundant compounds in acetonic A. Arabica aerial parts extracts (Table 5).
Overall, GC/MS chromatogram revealed that the 9-Octadecenamide, (Z)- (41.656% and 61.097%) and Hexadecanamide (36.542% and 20.238%) were the most abundant compounds for ethanolic and acetonic A. aleppica aerial parts extracts, respectively. Whereas, 9-Octadecenamide, (Z)- (41.280% and 53.990%) and Hexadecanamide (30.828% and 14.445%) were the most abundant compounds for ethanolic and acetonic A. Arabica aerial parts extracts, respectively.
It has been reported the presence of camphor (930%), 1,8-cineole (9-42%), p-cymene (5-27%) and piperitone (3-50%) as a main components in A.
biebersteinii EOs (Toncer et al. 2010; Tabanca et al. 2011). Whereas, a-terpinen (41.42%), 2-carene (13.96%), m-cymene (13.41%) and 1,8-cineole (8.91%) were mainly presented in A. biebersteinii EOs using GC/MS analysis (Dehghan and Elmi 2014). While, Al-Said et al. (2016) reported 44 compounds in A. biebersteinii EOs; of which a-Terpinene (29.2%), p-Cymene (22.9%), Terpinen-4-ol (4.7%) and 1,8-Cineole (4.3%) were mainly detected in their EOs using GC/MS analysis. Moreover, Sevindiki et al. (2018) reported 29 components in A. biebersteinii EOs, of which 1,8-cineole (20.36%), cyclohexanone (8.39%), 2-cyclohexen-1-one (5.38%) and spathulenol (4.19%) were presented as a main components using GC/MS analysis.
Whereas, camphor (33-34%), 1,8-cineole (20-26%), p-cymene (14%), a-pinene (4%), a-terpineol (9%), a-bisabolol oxide (4%), T-cadinol (4%), caryophyllene oxide (3%) and spathulenol (3%) as a main components in A. aleppica EOs (Toncer et al. 2010).
GC/MS analysis has been extensively also used to investigate of other Achillea species for their EOs composition. In this regards, Boskovic et al. (2005) reported that p-pinene in A. millefolium, T-cadinol in A. lingulata, 1,8-cineole in A. holosericea and camphor in A. clavennae were the main constituents in their EOs. Moreover, Astafyeva et al. (2018) reported that aldehydes (41.93%), alcohols (21.24%), hydrocarbons (14.45%), aromatic hydrocarbons (7.78%), esters (3.21%) and ketones (2.37%) functional groups, were mainly recorded in the aqueous ethanolic (40 % v/v) vegetative parts A. micrantha extract. Whereas, Hatem et al. (2018) reported 51 compounds of which artemisia ketone (29.97%), a-thujone (13.34%), germacrene (11.5%) followed by a-cubebene (6.25%), spathulenol (3.63%), p-sesquiphellandrene (3.52%) and y-muurolene (3.27%) were mainly components in the fresh aerial parts of A. fragrantissima EOs. Moreover, Albayrak and Silahtarlioglu (2019) reported 45 compounds were presented in A. coarctata EOs; of which Camphor (29.44%), 1,8-cineole (19.87%), borneole (8.25%), p-eudesmol (7.65%) and caryophyllene oxide (7.29%) were mainly occurred using GC/MS analysis. Whereas, other compounds were presented in minor amounts (0.17%-2.91%).
Whereas, Dehghan and Elmi (2014) reported 20 compounds in A. millefolium EOs of which 1,8-cineole (28.0%), camphor (19.2%), borneol (98.8%) and p-pinene (6.3%) were mainly presented. While, 23 compounds in A. wilhelmsii EOs of which carvacrol (29.2%), linalool (10.3%), 1,8-cineole (11.0%), (E)-nerolidol (8.4%) and borneol (5.04%) were mainly presented in their EOs. Whereas, Farajpour ett al. (2017) reported that the 1,8-Cineole, 1.2-19.8%; p-thujone, 0.4-55.3%; camphor, 0.6-25.5%; germacrene-D, 2-20.6%; trans-nerolidol, 0.4-48.1%; isospathulenol, 0.5-36%; and cubenol, 0.1-42.9% were mainly detected in the A. millefolium EOs. Recently, Yener et al. (2020) reported that a-terpinene, p-eudesmol, piperitone, endo-borneol, artemisia ketone, verbenol, eucalyptol and camphor were the main constituents in Achillea species EOs.
In the current study, caryophyllene oxide was
recorded to be 0.264% in acetonic A. aleppica aerial parts extract; whereas, it was recorded to be 1.01% in A. biebersteinii EOs (Baris et al. 2006); 3% in A. aleppica EOs (Toncer et al. 2010); 2% in A. tenuifolia EOs (Manayi et al. 2014); 0.52% in A. fragrantissima EOs (Choucry 2017) and ranged between 0.76-11.9% in A. millefolium EOs (Farajpour et al. 2017). Otherwise, in the current study, oleic acid was recorded to be 41.656% and 61.097% for ethanolic and acetonic A. aleppica aerial parts extracts, respectively and to be 41.280% and 53.990% for ethanolic and acetonic A. Arabica aerial parts extract's, respectively. Whereas, it was 9.7% in A. tenuifolia EOs (Manayi et al. 2014). These differences could be attributed to the fact that geographical distribution, species, plant phynological stages and extraction type (solvents or EOs) affect Achillea sp. chemical composition (Dehghan and Elmi 2014; Al-Said et al. 2016; Farajpour et al. 2017).
Table 1. Collection sites of A. aleppica and A.arabica species.
Species_Collection site_Code_Altitude (m)_Annual rainfall (mm)
A. aleppica Damascus A.A 950 260
A. arabica Homs A.R 265 400
Table 2. GC/MS analysis of ethanolic A. aleppica aerial parts extracts.
Peak No RT (min) Name of Compound Peak area (%)
1 9.482 Eucalyptol 1.002
2 11.745 endo-Borneol 1.523
3 20.611 Hexadecanenitrile 0.748
4 22.259 Pentadecanal 2.027
5 24.126 Dodecanoic acid, 3-hydroxy- 3.314
6 24.982 Hexadecanamide 36.542
7 29.404 9-Octadecenamide, (Z)- 41.656
8 30.084 Tetradecanamide 9.965
9 32.232 9-Octadecenenitrile, (Z)- 0.803
10 33.743 13-Docosenamide, (Z)- 1.431
11 33.881 9-Octadecenoic acid (Z)-, tetradecyl ester 0.988
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Table 3. GC/MS analysis of acetonic A. aleppica aerial parts extracts.
Peak No RT (min) Name of Compound Peak area (%)
1 7.038 Styrene 0.142
2 9.491 Eucalyptol 0.777
3 10.087 p-Menth-8-en-1-ol, stereoisomer 0.260
4 10.599 Cyclohexanol, 1-methyl-4-(1-methylethenyl)-,cis 0.383
5 10.674 Thujone 0.428
6 11.749 endo-Borneol 0.689
7 17.412 Caryophyllene oxide 0.264
8 18.430 Eicosane 0.325
9 18.510 Nonadecane 0.832
10 18.596 Heptanoic acid, heptyl ester 0.263
11 19.693 Tetradecanal 0.608
12 20.199 Benzaldehyde, 4-(dimethylamino)- 0.183
13 20.622 Octadecanal 1.170
14 20.885 Dodecyl nonyl ether 0.803
15 21.381 10-Octadecenal 0.833
16 22.270 Pentadecanal 1.676
17 23.211 Z,Z-4,16-Octadecadien-1-ol acetate 1.016
18 23.621 9-Hexadecenoic acid 0.549
19 24.994 Hexadecanamide 20.238
20 25.500 Palmitoleic acid 0.627
21 29.434 9-Octadecenamide, (Z)- 61.097
22 30.101 Nonadecanamide 5.601
23 32.240 9-Octadecenenitrile, (Z) 1.238
Table 4. GC/MS analysis of ethanolic A.arabica aerial parts extracts.
Peak No RT (min) Name of Compound Peak area (%)
1 13.654 4-Hydroxy-a-Thujone 5.002
2 14.894 4-Hydroxy-ß-Thujone 5.913
3 20.618 Dodecanoic acid, 3-hydroxy- 0.925
4 24.124 Palmitoleamide 5.608
5 24.984 Hexadecanamide 30.828
6 27.163 Humulenol-ll 1.505
7 29.405 9-Octadecenamide, (Z)- 41.280
8 30.088 Dodecanamide 8.940
Table 5. GC/MS analysis of acetonic A.arabica aerial parts extracts.
Peak No RT (min) Name of Compound Peak area (%)
1 7.038 Styrene 0.375
2 9.322 Benzoic acid, 2,4-dimethyl-, (2,4-dimethylpheny)methyl ester 0.145
3 11.866 Octanoic acid, 7-oxo- 0.358
4 12.724 Isopulegol acetate 0.599
5 13.430 1,4-dihydroxy-p-menth-2-ene 0.269
6 14.415 Dimethylmuconic acid 0.082
7 14.512 Ricinoleic acid 0.170
8 14.712 Arginine 0.160
9 14.896 1,6-Octadiene, 3-ethoxy-3,7-dimethyl- 1.526
10 17.201 1-Eicosanol 0.080
11 18.508 Tetradecane 0.559
12 18.836 Benzeneacetic acid, 4-tetredecyl ester 0.191
13 19.185 Cyclobuta[a]dibenzo[c.f]cycloheptadiene, 7-oxo- 0.847
14 19.677 Tetradecanal 0.426
15 20.197 Didodecyl phthalate 0.222
16 20.620 Hexadecanenitrile 0.744
17 20.887 Nonadecane 0.580
18 21.373 trans-2-Hexadecenoic acid 0.835
19 22.265 Tridecanal 1.007
20 23.200 9-Octadecenenitrile, (Z) 0.691
21 23.604 Hexadecanal 0.350
22 24.162 13-Docosenamide, (Z)- 7.829
23 24.582 9-Hexadecenoic acid 0.680
24 24.992 Hexadecanamide 14.445
25 25.502 Octadecanenitrile 0.606
26 27.156 Phthaloylaspartic acid 0.506
27 27.648 a-Amyrin 0.327
28 29.427 9-Octadecenamide, (Z)- 53.990
29 30.095 Deoxyspergualin 5.003
30 31.556 4-((2-Amino-phenylthio)-1-benzyl-6-methylpiperidin-2-thione 0.933
31 32.238 Palmitoleonitrile 1.011
32 33.344 Carnegine 1.276
33 33.628 Benzen,1,1-[2-methyl-2-(phenylthio)cycloprppylidene]bis- 1.505
34 33.748 Cis-11-Eicosenamide 1.668
CONCLUSION
Ethanolic and acetonic A. aleppica and A. arabica aerial parts extracts have been investigated for their chemical composition using GC/MS analysis. Among different compounds detected in GC/MS ethanolic and acetonic A. Arabica and A. aleppica aerial parts extracts,
9-Octadecenamide, (Z)- and Hexadecanamide were the most abundant compounds. The two studied Achillea species showed some differences in their chemical composition of their extracts. The observed differences in their extracts composition could be attributed to the tested solvents, studied Achillea species and their geographical distribution.
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ACKNOWLEDGMENT
I thank I. Othman (Director General of AECS) and N. MirAli (Head of Molecular Biology and Biotechnology
The authors declare that they have no potential
conflicts of interest.
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