Phytochemical analysis of two Achillea (Asteraceae) species using GC/MS technique

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 (Stojanović et al . 2005; Toncer et al . 2010; Tabanca et al . 2011; Albayrak and Silahtarlıoğlu 2019); antioxidant (Toncer et al . 2010; Manayi et al . 2012; Polatoglu et al . 2013; Albayrak and Silahtarlıoğlu 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 Silahtarlıoğlu 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, antiinflammatory 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; Başer 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 Silahtarlıoğlu 2019) and aqueous ethanolic (40 % v/v) vegetative parts A. 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 MiddleSouthern 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 × 0.25 mm × 0.25 μm)], 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

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, α-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%), α-pinene (4%), α-terpineol (9%), α-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 β-pinene in A. millefolium , τ-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%), α-thujone (13.34%), germacrene (11.5%) followed by α-cubebene (6.25%), spathulenol (3.63%), β-sesquiphellandrene (3.52%) and γ-muurolene (3.27%) were mainly components in the fresh aerial parts of A. fragrantissima EOs. Moreover, Albayrak and Silahtarlıoğlu (2019) reported 45 compounds were presented in A. coarctata EOs; of which Camphor (29.44%), 1,8-cineole (19.87%), borneole (8.25%), β-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 β-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%; β-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 α-terpinene, β-eudesmol, piperitone, endoborneol, 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

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-α-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	α-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.

ACKNOWLEDGMENT

I thank I. Othman (Director General of AECS) and N. MirAli (Head of Molecular Biology and Biotechnology Department in AECS) for their support.

CONFLICTS OF INTEREST

The authors declare that they have no potential conflicts of interest.