As shown in Figure 2, comparison of the essential oil yield among different samples revealed that the highest content of essential oil belongs to the harvested aerial parts of S. limbata in the vegetative stage at an altitude of 1500 m (0.86% v/w) while no significant difference was observed in the essential oil content among other groups. In a published study authors observed the significant impact of different altitudes and phenological stages on the essential oil yield [22,23]. It was revealed that plant performance is strongly influenced by various factors such as altitude, climate, soil, developmental stages, extraction and analysis methods, genetic factors, abiotic stresses, and slope and modeling techniques can predict these factors in other areas [24,25,26,27, 28]. The results of the current study were compatible with other studies that found the highest content of essential oil of Origanum majorana in the vegetative stage, so they proposed the vegetative stage as the best stage to harvest Origanum majorana [29,30, 31,32, 33,34,35,36].Similar results were also found byon the essential oil content of Nepeta kotschyr [37]. Moreover, according to a study conducted by the highest yield of essential oil in Teucrium polium L. was obtained in the vegetative stage [38]. These results are in agreement with our findings. The accumulation of essential oil in vegetative stage could be due to the fact that plant protection is supplied by phenolic components which are in high amount in this stage [39].
On the contrary, it was reported bythat the highest essential oil in Satureja mutica is acquired in the flowering stage[40]. In another study [41] determined that the highest value of essential oil of Mentha pieperata in flowering stage, which contradicts our findings. Also, the percentage of essential oils in vegetative stage in Thymus vulgaris was the lowest and it rose in flowering stage [42]. One explanation for the increase in essential oil content in the flowering stage is the maintenance of the reproductive stage [43,44] and to attract insects for pollination [35]. Nevertheless, other researches have illustrated that the lowest amount of essential oils in the vegetative stage could be due to the lower activity of some enzymes in synthesizing phenolic compounds in this stage [45]. Since photosynthetic products accumulate in the endosperm during plant growth, it leads to a decrease in the amount of essential oil [46]. It is clear that phenological stages have a great impact on the essential oil metabolism, enzymatic activity and finally essential oil content [45].
As illustrated in Tables 1, 2 and 3, twenty-eight components were identified in the S. limbata essential oil by means of GC-FID and GC-MS analysis which represented about 96.5% to 99.7% of the total composition of the obtained essential oil. In the current study, the main identified compounds were α-pinene (14.7-38.7%), β-pinene (12.5-26.2%), allo-aromadendrene (9.2-21.7%), germacrene D (4.2-8.3%), bicyclogermacrene (6.5-14.5 %), and spathulenol (7.5-25.4 %). The molecular structures of the main identified compounds from S. limbata essential oil are presented in the Figure 3. Comparing the results of the current study to others, α-pinene (23.7 %), β-pinene (18.7%), sabinene (14.5%), 1, 8-cineole (9.9%) and β-caryophyllene (7.1%) as the major components of S. limbata essential oil in the flowering stage [47]. In another research, following GC-MS analysis of the aerial parts of S. limbata obtained from Turkey, 42 components were characterized representing 95.6% to 98.1% of the compounds including α-pinene (11.2-24.3%), β-pinene (10.0-20.9%) and sabinene (14.6-17.4%) as the major constituents of the essential oil [48].
Comparing of the monoterpenes and sesquiterpenes contents of the S. limbata essential oil at different altitudes and phenological stages in Figure 4, the amount of monoterpenes has decreased from vegetative stage to seed ripening stage; however, the obtained results for sesquiterpenes were reverse. These findings for Artemisia herba-alba essential oil were previously observed in another study [49]. Moreover, during the developing plants the amount of sesquiterpenes increased in Cannabis sativa L. which are in line with our results[50]. As we found out in our research, the highest amount of monoterpenes was related to the vegetative period at 2000 m, while the highest amount of sesquiterpenes was obtained in seed ripening stage at altitudes of 1500 and 2500 m. [51] 2002 in a research on Thymus vulgaris at different growth stages confirmed that the highest content of the monoterpene was related to the vegetative stage.
As shown in Figure 5, the content of α-pinene, β-pinene, alloaromadendrene, germacrene D, bicyclogermacrene, and spathulenol illustrates some changes in different altitudes and developmental stages. The highest percentage of monoterpenes including α-pinene (41.3%) and β-pinene (30.1%) was obtained in the vegetative stage at 2000 m. The contents of α-pinene and β-pinene were decreased to the lowest values in the ripening stage. Moreover, the highest content for alloaromadendrene was measured 20.6% and 20.7% in the ripening stage at 1500 m and 2500 m, respectively without any significant difference between them. However, the lowest quantity was obtained 3.5% for the vegetative stage at 2000 m. The most abundant germacrene D reached in ripening stage at 2500 m (8.3%) and the lowest amount (1.2%) achieved at 2000 m in the vegetative stage. Moreover, high value of bicyclogermacrene was attained in the ripening stage at 1000 m and 2000m (14.5% and 14.3%, respectively) while no significant difference was observed between the mentioned altitudes. On the contrary, the lowest amount was attained in the vegetative stage at 1500 m (4.2%). Furthermore, the highest and the lowest contents of spathulenol (25.4% and 7.2%) were gained in the ripening stage at 2500 m and vegetative stage at 2000 m, respectively. Variation in the percentage of compounds at different stages of phenology and altitudes could be due to the high or low synthesis of compounds by enzymes, which leads to different percentages of compounds in essential oils.
Table 1. The percentage of chemical compositions of Salvia limbata essential oil in the vegetative stage at different altitudes
No.
|
Compounds
|
RI
|
1500 m
|
2000 m
|
2500 m
|
1
|
α-Thujene
|
922
|
Tr
|
Tr
|
Tr
|
2
|
α-Pinene
|
938
|
30.4±0.1
|
41.3±0.2
|
28.5±0.8
|
3
|
Camphene
|
952
|
1.2±0.1
|
1.4±0.2
|
0.5±0.0
|
4
|
Sabinene
|
975
|
2.4±0.1
|
1.9±0.1
|
4.1±0.1
|
5
|
β-Pinene
|
980
|
25.4±0.3
|
30.1±0.1
|
24.2±0.2
|
6
|
Myrcene
|
985
|
Tr
|
Tr
|
Tr
|
7
|
p-Cymene
|
1025
|
Tr
|
Tr
|
Tr
|
8
|
Limonene
|
1029
|
0.9±0.1
|
0.6±0.04
|
0.4±0.0
|
9
|
Z-β-Ocimene
|
1035
|
Tr
|
Tr
|
Tr
|
10
|
Linalool
|
1085
|
Tr
|
Tr
|
Tr
|
11
|
α-Campholenal
|
1103
|
Tr
|
Tr
|
Tr
|
12
|
Trans-Pinocarveol
|
1125
|
Tr
|
0.2±0.0
|
Tr
|
13
|
Trans-Verbenol
|
1162
|
Tr
|
0.3±0.0
|
0.2±0.01
|
14
|
Borneol
|
1186
|
Tr
|
0.2±0.0
|
Tr
|
15
|
Terpine-4-ol
|
1203
|
Tr
|
0.4±0.0
|
0.2±0.0
|
16
|
Myrtenal
|
1216
|
Tr
|
0.4±0.0
|
0.2±0.0
|
17
|
Verbenone
|
1239
|
0.3±0.1
|
0.2±0.0
|
0.3±0.0
|
18
|
Bornyl-acetate
|
1316
|
1±0.1
|
Tr
|
Tr
|
19
|
Eugenol
|
1340
|
Tr
|
0.9±0.1
|
1.4±0.2
|
20
|
β-Caryophyllene
|
1426
|
Tr
|
Tr
|
0.2±0.0
|
21
|
Allo-Aromadendrene
|
1482
|
9.5±0.1
|
3.5±0.2
|
12.6±0.3
|
22
|
γ-Muurolene
|
1485
|
Tr
|
0.6±0.0
|
Tr
|
23
|
Germacrene D
|
1498
|
3.7±0.2
|
1.2±0.1
|
4.1±0.2
|
24
|
Bicyclogermacrene
|
1505
|
4.6±0.2
|
5.5±0.3
|
8.5±0.9
|
25
|
Eugenol-acetate
|
1521
|
Tr
|
0.6±0.0
|
Tr
|
26
|
Spathulenol
|
1575
|
12.6±0.1
|
7.2±0.2
|
13.8±0.2
|
27
|
Caryophyllene oxide
|
1580
|
4.3±0.1
|
Tr
|
Tr
|
28
|
Sclareol
|
2200
|
1.9±0.1
|
0.2±0.0
|
0.5±0.0
|
|
Monoterpene hydrocarbons
|
|
60.3
|
75.3
|
57.7
|
|
Oxygenated monoterpenes
|
|
1.3
|
2.6
|
2.3
|
|
Sesquiterpene hydrocarbons
|
|
17.8
|
10.8
|
25.4
|
|
Oxygenated sesquiterpenes
|
|
18.8
|
8.0
|
14.3
|
|
Total
|
|
98.2
|
96.7
|
99.7
|
Data are presented as mean±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace (<0.1%)
Table 2. The percentage of chemical compositions of Salvia limbata essential oil in the flowering stage at different altitudes
No.
|
Compounds
|
RI
|
1500 m
|
2000 m
|
2500 m
|
1
|
α-Thujene
|
922
|
Tr
|
Tr
|
Tr
|
2
|
α-Pinene
|
938
|
21.2±0.2
|
29.5±0.4
|
21.7±0.6
|
3
|
Camphene
|
952
|
1.3±0.1
|
1.4±0.2
|
1.8±0.2
|
4
|
Sabinene
|
975
|
1.7±0.1
|
3.3±0.1
|
1.8±0.1
|
5
|
β-Pinene
|
980
|
17.3±0.1
|
26.2±0.2
|
20.1±0.2
|
6
|
Myrcene
|
985
|
Tr
|
Tr
|
Tr
|
7
|
p-Cymene
|
1025
|
Tr
|
Tr
|
Tr
|
8
|
Limonene
|
1029
|
1±0.1
|
1±0.1
|
0.8±0.1
|
9
|
Z-β-Ocimene
|
1035
|
Tr
|
1.2±0.1
|
Tr
|
10
|
Linalool
|
1085
|
Tr
|
Tr
|
Tr
|
11
|
α-Campholenal
|
1103
|
Tr
|
Tr
|
Tr
|
12
|
Trans-Pinocarveol
|
1125
|
0.3±0.0
|
Tr
|
0.4±0.0
|
13
|
Trans-Verbenol
|
1162
|
Tr
|
Tr
|
0.3±0.0
|
14
|
Borneol
|
1186
|
Tr
|
Tr
|
0.3±0.0
|
15
|
Terpine-4-ol
|
1203
|
0.6±0.0
|
0.6±0.0
|
Tr
|
16
|
Myrtenal
|
1216
|
0.6±0.0
|
0.6±0.1
|
Tr
|
17
|
Verbenone
|
1239
|
0.2±0.0
|
0.3±0.0
|
Tr
|
18
|
Bornyl-acetate
|
1316
|
Tr
|
0.2±0.0
|
Tr
|
19
|
Eugenol
|
1340
|
1.2±0.1
|
0.4±0.0
|
0.5±0.0
|
20
|
β-Caryophyllene
|
1426
|
Tr
|
Tr
|
Tr
|
21
|
Allo-Aromadendrene
|
1482
|
15.2±0.2
|
9.2±0.1
|
14.6±0.2
|
22
|
γ-Muurolene
|
1485
|
0.5±0.0
|
3.2±0.1
|
0.3±0.0
|
23
|
Germacrene D
|
1498
|
5.7±0.2
|
4.4±0.1
|
4.2±0.2
|
24
|
Bicyclogermacrene
|
1505
|
10.7±0.2
|
6.5±0.1
|
13.6±0.2
|
25
|
Eugenol-acetate
|
1521
|
Tr
|
Tr
|
Tr
|
26
|
Spathulenol
|
1575
|
18.6±0.3
|
7.5±0.1
|
15.5±0.3
|
27
|
Caryophyllene oxide
|
1580
|
1±0.1
|
2.1±0.2
|
Tr
|
28
|
Sclareol
|
2200
|
1.7±0.2
|
Tr
|
0.4±0.0
|
|
Monoterpene hydrocarbons
|
|
42.5
|
62.6
|
46.2
|
|
Oxygenated monoterpenes
|
|
2.9
|
2.1
|
1.5
|
|
Sesquiterpene hydrocarbons
|
|
32.1
|
23.3
|
32.7
|
|
Oxygenated sesquiterpenes
|
|
21.3
|
9.6
|
15.9
|
|
Total
|
|
98.8
|
97.6
|
96.3
|
Data are presented as mean±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace (<0.1%).
Table 3. The percentage of chemical compositions of Salvia limbata essential oil in the ripening stage at different altitudes
No.
|
Compounds
|
RI
|
1500 m
|
2000 m
|
2500 m
|
1
|
α-Thujene
|
922
|
Tr
|
Tr
|
Tr
|
2
|
α-Pinene
|
938
|
14.9±0.2
|
19.5±0.2
|
14.7±0.1
|
3
|
Camphene
|
952
|
0.9±0.0
|
1.6±0.2
|
0.6±0.0
|
4
|
Sabinene
|
975
|
1.3±0.2
|
2.2±0.2
|
1.1±0.1
|
5
|
β-Pinene
|
980
|
14.2±0.3
|
17.6±0.3
|
12.5±0.1
|
6
|
Myrcene
|
985
|
Tr
|
Tr
|
Tr
|
7
|
p-Cymene
|
1025
|
Tr
|
Tr
|
Tr
|
8
|
Limonene
|
1029
|
0.5±0.0
|
0.7±0.0
|
0.4±0.0
|
9
|
Z-β-Ocimene
|
1035
|
Tr
|
Tr
|
Tr
|
10
|
Linalool
|
1085
|
Tr
|
Tr
|
Tr
|
11
|
α-Campholenal
|
1103
|
Tr
|
Tr
|
Tr
|
12
|
Trans-Pinocarveol
|
1125
|
Tr
|
Tr
|
Tr
|
13
|
Trans-Verbenol
|
1162
|
Tr
|
0.2±0.0
|
Tr
|
14
|
Borneol
|
1186
|
Tr
|
Tr
|
Tr
|
15
|
Terpine-4-ol
|
1203
|
0.3±0.0
|
0.5±0.0
|
Tr
|
16
|
Myrtenal
|
1216
|
0.3±0.0
|
0.5±0.0
|
Tr
|
17
|
Verbenone
|
1239
|
0.2±0.0
|
0.2±0.0
|
Tr
|
18
|
Bornyl-acetate
|
1316
|
Tr
|
Tr
|
Tr
|
19
|
Eugenol
|
1340
|
0.6±0.0
|
Tr
|
1.5±0.2
|
20
|
β-Caryophyllene
|
1426
|
Tr
|
2.6±0.0
|
Tr
|
21
|
Allo-Aromadendrene
|
1482
|
20.6±0.2
|
16.3±0.2
|
21.7±0.2
|
22
|
γ-Muurolene
|
1485
|
0.3±0.0
|
0.5±0.0
|
Tr
|
23
|
Germacrene D
|
1498
|
7.6±0.2
|
6.3±0.2
|
8.3±0.3
|
24
|
Bicyclogermacrene
|
1505
|
14.5±0.0
|
14.3±0.2
|
12.4±0.1
|
25
|
Eugenol-acetate
|
1521
|
Tr
|
0.9±0.0
|
Tr
|
26
|
Spathulenol
|
1575
|
22.4±0.2
|
12.6±0.1
|
25.4±0.2
|
27
|
Caryophyllene oxide
|
1580
|
Tr
|
Tr
|
Tr
|
28
|
Sclareol
|
2200
|
0.7±0.0
|
Tr
|
0.4±0.0
|
|
Monoterpene hydrocarbons
|
|
31.8
|
41.6
|
29.3
|
|
Oxygenated monoterpenes
|
|
1.4
|
1.4
|
1.5
|
|
Sesquiterpene hydrocarbons
|
|
43.0
|
40.0
|
42.4
|
|
Oxygenated sesquiterpenes
|
|
23.1
|
13.5
|
25.8
|
|
Total
|
|
99.3
|
96.5
|
99.0
|
Data are presented as mean±SD; RI indicates retention indices relative to C6-C24 n-alkanes; Tr indicates trace