# GC Analyses ofSalvia Seeds as Valuable Essential Oil Source

Authors: Mouna Ben Taârit; Kamel Msaada; Karim Hosni; Brahim Marzouk
Journal: Advances in Chemistry
 (2014)

---

## Abstract

The essential oils of seeds ofSalvia verbenaca, Salvia officinalis, and Salvia sclarea were obtained by hydrodistillation and analyzed by gas chromatography (GC) and GC-mass spectrometry. The oil yields (w/w) were 0.050, 0.047, and 0.045% in S. verbenaca, S. sclarea, and S. officinalis, respectively. Seventy-five compounds were identified. The essential oil composition of S. verbenaca seeds showed that over 57% of the detected compounds were oxygenated monoterpenes followed by sesquiterpenes (24.04%) and  labdane type diterpenes (5.61%). The main essential oil constituents were camphor (38.94%), caryophyllene oxide (7.28%), and 13-epi-manool (5.61%), while those of essential oil of S. officinalis were α-thujone (14.77%), camphor (13.08%), and 1,8-cineole (6.66%). In samples of S. sclarea, essential oil consists mainly of linalool (24.25%), α-thujene (7.48%), linalyl acetate (6.90%), germacrene-D (5.88%), bicyclogermacrene (4.29%), and α-copaene (4.08%). This variability leads to a large range of naturally occurring volatile compounds with valuable industrial and pharmaceutical outlets.


---

## Body

## 1. Introduction

The genusSalvia (Lamiaceae) comprises nearly 900 species widely spread throughout the world, which display marked morphological and genetic variations according to their geographical origin [1]. SeveralSalviaspecies, namely,Salvia officinalis,Salvia sclarea, andSalvia verbenaca, are widely used in folk medicine [2]. Potential therapeutic activities of theseSalvia species are due to their essential oils [3], since these species are known to possess antioxidant, antimicrobial, antifungal, and aromatic properties [4]. Chemical composition of essential oils reveals differences among theseSalvia species [5–7]. Numerous investigations onSalvia officinalis show that 1,8-cineole, α-thujone, β-thujone, and camphor are the main compounds of the essential oil [8–10]. Linalool, linalyl acetate, and germacrene-D characterizeS. sclareaplants [11].Salvia species also display great intraspecific essential oil variations according to geographical origin, since sabinene, cadinene, terpinen-4-ol, and pinene are shown to be typical compounds ofS. verbenaca essential oil originated from Saudi Arabia [4], while β-phellandrene and (E)-caryophyllene prevail in essential oil from Greece [7]. In Tunisia,S. verbenacaessential oil shows variations of composition according to the region origin [12, 13] and in respect to the studied plant part [14].These numerous studies are focused on aerial parts of these species, while works interested in seeds are scanty in spite of their interest. In fact,Salvia seeds provide dietary and healthy oil rich in essential fatty acids (linolenic and linoleic acids) [12, 15, 16] that promote decrease in coronary heart diseases [17]. Besides, the seeds ofSalvia species often produce mucilage on wetting [18]. This mucilage is used for lacquerware [19]. In eastern countries, the mucilage is used for the treatment of eye diseases [20].In our continuing research on essential oil with pharmacological potential and food industry applications, we report in this paper chemical analysis ofS. verbenaca,S. sclarea, andS. officinalis seeds as a new valuable essential oil source.


## 2. Materials and Methods

### 2.1. Plant Material

The seeds ofSalvia verbenaca (accession PI 420430) were originated from Spain;Salvia officinalis(accession W6 20659) andSalvia sclarea(accession W6 20660) which are from Italy were kindly supplied by the US National Plant Germplasm System (NPGS).


### 2.2. Essential Oil Isolation

The seeds of each species were ground in a mortar before essential oil isolation [12] and were subjected to conventional hydrodistillation for 90 min followed by a liquid-liquid extraction using diethyl ether and n-pentane mixture (v/v) as solvent. The concentration step was carried out at 35°C using a Vigreux column and the essential oils obtained were dried over anhydrous sodium sulphate and stored in amber vials at −18°C until they were analyzed.


## 2.1. Plant Material

The seeds ofSalvia verbenaca (accession PI 420430) were originated from Spain;Salvia officinalis(accession W6 20659) andSalvia sclarea(accession W6 20660) which are from Italy were kindly supplied by the US National Plant Germplasm System (NPGS).


## 2.2. Essential Oil Isolation

The seeds of each species were ground in a mortar before essential oil isolation [12] and were subjected to conventional hydrodistillation for 90 min followed by a liquid-liquid extraction using diethyl ether and n-pentane mixture (v/v) as solvent. The concentration step was carried out at 35°C using a Vigreux column and the essential oils obtained were dried over anhydrous sodium sulphate and stored in amber vials at −18°C until they were analyzed.


## 3. Chromatographic Analysis

### 3.1. Gas Chromatography (GC-FID)

The essential oils were analysed by gas chromatography using a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar HP Innowax (PEG) column and an apolar HP-5 column (30 m × 0.25 mm, 0.25μm film thicknesses) were used. The carrier gas was N2 with a flow rate of 1.6 mL/min; split ratio was 60 : 1. The analysis was performed using the following temperature program: oven temps isotherm at 35°C for 10 min, from 35 to 205°C, at the rate of 3°C/min, and isotherm at 205°C during 10 min. Injector and detector temperatures were held, respectively, at 250 and 300°C. The volume injected was 1 μL.


### 3.2. Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer with electron impact ionization (70 eV). A HP-5MS capillary column (30 m × 0.25 mm, 0.25μm film thickness) was used. The column temperature was programmed from 50°C to rise to 240°C at a rate of 5°C/min. The carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was 60 : 1. Scan time and mass range were 1 s and 40–300 m/z, respectively.


### 3.3. Compounds Identification

The identification of the essential oil constituents was based on the comparison of their retention indexes relative to (C8–C22) n-alkanes with those of literature or with those of authentic compounds available in our laboratory. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC-MS data system and other published mass spectra [21].


### 3.4. Statistical Analyses

Data were subjected to statistical analysis using “Statistica” statistical program package [22]. The percentages of volatile compounds are means of three experiments; the one-way analysis of variance (ANOVA) followed by Duncan multiple range test was employed and the differences between individual means were deemed to be significant at P<0.05.


## 3.1. Gas Chromatography (GC-FID)

The essential oils were analysed by gas chromatography using a Hewlett-Packard 6890 gas chromatograph (Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and an electronic pressure control (EPC) injector. A polar HP Innowax (PEG) column and an apolar HP-5 column (30 m × 0.25 mm, 0.25μm film thicknesses) were used. The carrier gas was N2 with a flow rate of 1.6 mL/min; split ratio was 60 : 1. The analysis was performed using the following temperature program: oven temps isotherm at 35°C for 10 min, from 35 to 205°C, at the rate of 3°C/min, and isotherm at 205°C during 10 min. Injector and detector temperatures were held, respectively, at 250 and 300°C. The volume injected was 1 μL.


## 3.2. Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed on a gas chromatograph HP 5890 (II) interfaced with a HP 5972 mass spectrometer with electron impact ionization (70 eV). A HP-5MS capillary column (30 m × 0.25 mm, 0.25μm film thickness) was used. The column temperature was programmed from 50°C to rise to 240°C at a rate of 5°C/min. The carrier gas was helium with a flow rate of 1.2 mL/min; split ratio was 60 : 1. Scan time and mass range were 1 s and 40–300 m/z, respectively.


## 3.3. Compounds Identification

The identification of the essential oil constituents was based on the comparison of their retention indexes relative to (C8–C22) n-alkanes with those of literature or with those of authentic compounds available in our laboratory. Further identification was made by matching their recorded mass spectra with those stored in the Wiley/NBS mass spectral library of the GC-MS data system and other published mass spectra [21].


## 3.4. Statistical Analyses

Data were subjected to statistical analysis using “Statistica” statistical program package [22]. The percentages of volatile compounds are means of three experiments; the one-way analysis of variance (ANOVA) followed by Duncan multiple range test was employed and the differences between individual means were deemed to be significant at P<0.05.


## 4. Results

Hydrodistillation of full ripened seeds ofS. verbenaca,S. sclarea, andS. officinalisoffered essential oils with average yields of 0.050, 0.047, and 0.045% (w/w on the dry weight basis), respectively.Essential oil constituents ofSalvia seeds were presented in Table 1. The results of analysis of essential oil ofS. verbenacaseeds by GC and GC-MS techniques revealed the occurrence of thirty-two compounds. The essential oil composition showed that over 57% of the detected compounds were oxygenated monoterpenes followed by sesquiterpenes (24.04%) and labdane type diterpenes (5.61%). Apart from camphor, the main essential oil constituents of this sample were caryophyllene oxide (7.28%), 13-epi-manool (5.61%), δ-elemene (3.97%), and β-eudesmol (3.76%).Table 1
Essential oil composition (%w/w) ofSalvia verbenaca(Sv), Salvia officinalis (So), and Salvia sclarea (Ss) seeds.
Number
Compounds*
RIa
RIb
Sv
So
Ss
Identification
1
Hexanal
800
1093
0.42
—
—
GC-MS, Co-GC
2
(E)-2-Hexenal
852
1232
—
—
0.03
GC-MS, Co-GC
3
1-Hexanol
878
1360
—
1.29
—
GC-MS, Co-GC
4
Tricyclene
927
1014
0.96a
0.23b
0.08c
GC-MS
5
α-Thujene
930
1035
—
3.08b
7.48a
GC-MS
6
α-Pinene
939
1032
0.44b
1.26a
0.27c
GC-MS
7
δ-3-Carene
1012
1159
0.50a
0.03b
—
GC-MS
8
p-Cymene
1022
1280
0.37b
1.52a
0.12c
GC-MS
9
Limonene
1030
1203
—
—
0.17
GC-MS, Co-GC
10
β-Phellandrene
1030
1218
—
—
0.03
GC-MS
11
1,8-Cineole
1033
1213
—
6.66a
0.12b
GC-MS, Co-GC
12
cis-Sabinene hydrate
1068
1556
—
0.19
—
GC-MS
13
cis-Linalool oxide
1072
1478
—
0.13
—
GC-MS
14
α-Fenchone
1087
1406
0.26
—
—
GC-MS
15
trans-Linalool oxide
1087
1450
1.05
—
—
GC-MS
16
Linalool
1098
1553
0.84b
0.68b
24.25a
GC-MS, Co-GC
17
n-Undecane
1100
1100
2.65a
0.48c
0.75b
GC-MS, Co-GC
18
α-Thujone
1102
1429
0.52b
14.77a
—
GC-MS
19
cis-allo-Ocimene
1113
1382
1.55a
0.14b
1.46b
GC-MS
20
β-Thujone
1114
1451
—
4.30
—
GC-MS
21
Geijerene
1140
1338
—
1.01
—
GC-MS
22
Camphor
1143
1532
38.94a
13.08b
—
GC-MS
23
Borneol
1165
1719
—
3.54
—
GC-MS
24
Terpinen-4-ol
1177
1611
0.88a
0.09c
0.37b
GC-MS
25
p-Cymen-8-ol
1185
1864
—
0.17
—
GC-MS
26
δ-Terpineol
1187
1682
0.49b
2.42a
0.20c
GC-MS
27
α-Terpineol
1189
1706
2.03a
0.91b
0.20c
GC-MS
28
Myrtenal
1190
1648
—
0.55
—
GC-MS
29
Myrtenol
1194
1804
—
0.28
—
GC-MS
30
cis-Sabinol
1210
1800
—
0.18
—
GC-MS
31
Nerol
1228
1797
—
—
0.98
GC-MS, Co-GC
32
Linalyl acetate
1239
1565
2.53b
0.08c
6.90a
GC-MS
33
Geraniol
1254
1857
—
0.33b
2.79a
GC-MS, Co-GC
34
Bornyl acetate
1285
1597
0.79a
0.16b
—
GC-MS
35
Thymol
1293
2198
—
0.37a
0.10b
GC-MS
36
Carvacrol
1296
nd
—
0.83b
1.93a
GC-MS
37
δ-Elemene
1337
1479
3.97a
0.07c
0.85b
GC-MS
38
α-Cubebene
1348
1456
—
—
2.86
GC-MS
39
α-Terpinyl acetate
1353
1709
4.77a
1.81b
0.29c
GC-MS
40
Eugenol
1357
2192
—
0.83
—
GC-MS, Co-GC
41
Neryl acetate
1366
1733
2.40a
—
0.36b
GC-MS, Co-GC
42
α-Ylangene
1372
1493
—
0.04b
0.24a
GC-MS
43
α-Copaene
1376
1497
—
0.01b
4.08a
GC-MS
44
β-Damascenone
1381
1838
—
0.06
—
GC-MS
45
Geranyl acetate
1383
1765
—
—
1.94
GC-MS, Co-GC
46
β-Bourbonene
1384
1533
1.73a
—
0.35b
GC-MS
47
β-Elemene
1389
1600
—
0.16
—
GC-MS
48
Methyl eugenol
1402
2030
—
0.18
—
GC-MS, Co-GC
49
β-Caryophyllene
1413
1612
0.27a
0.19b
0.24a
GC-MS
50
β-Cubebene
1419
1549
1.50a
—
0.72b
GC-MS
51
Aromadendrene
1439
1628
0.66a
0.18b
—
GC-MS
52
(Z)-β-Farnesene
1441
1668
1.76
—
—
GC-MS
53
α-Humulene
1454
1687
0.67b
3.71a
0.08c
GC-MS, Co-GC
54
allo-Aromadendrene
1460
1661
1.29b
1.43a
0.06c
GC-MS
55
α-Amorphene
1474
1680
—
0.47
—
GC-MS
56
γ-Muurolene
1477
1704
0.05
—
—
GC-MS
57
Germacrene-D
1479
1726
—
1.18b
5.88a
GC-MS
58
epi-Cubebol
1491
1900
—
0.34a
0.24b
GC-MS
59
Bicyclogermacrene
1494
1755
—
1.29b
4.29a
GC-MS
60
(E,E)-α-Farnesene
1506
1758
—
0.24
—
GC-MS
61
β-Bisabolene
1508
1741
1.10a
0.72b
—
GC-MS
62
γ-Cadinene
1513
1776
—
0.08
—
GC-MS
63
δ-Cadinene
1517
1773
—
0.53a
0.24b
GC-MS
64
α-Calacorene
1542
1942
—
—
0.10
GC-MS
65
Germacrene-B
1558
1854
—
0.08b
1.29a
GC-MS
66
(E)-Nerolidol
1563
2050
—
1.41
—
GC-MS
67
Spathulenol
1575
2144
—
0.08a
0.03b
GC-MS
68
Caryophyllene oxide
1579
2008
7.28a
0.16c
3.18b
GC-MS
69
Viridiflorol
1592
2104
—
2.66
—
GC-MS
70
Humulene epoxide I
1596
2045
—
—
1.55
GC-MS
71
Humulene epoxide II
1606
2071
—
0.25
—
GC-MS
72
T-Cadinol
1642
2187
—
0.15b
0.52a
GC-MS
73
α-Cadinol
1643
nd
—
0.41b
0.88a
GC-MS
74
β-Eudesmol
1649
nd
3.76a
0.47c
1.72b
GC-MS
75
13-epi-Manool
Nd
nd
5.61a
2.22b
0.59c
GC-MS
Compound classes
Alcohols
2.46a
0.42b
0.03c
Aliphatic hydrocarbons
Alkanes
—
1.29
—
Aldehydes
0.84a
0.48c
0.75b
Monoterpene hydrocarbons
3.82c
6.45b
9.60a
Oxygenated monoterpenes
57.30a
50.14b
38.38c
Sesquiterpene hydrocarbons
13.00b
11.39b
21.27a
Norisoprenoids with 13 carbons
—
0.06
—
Phenylpropanes
—
1.01
—
Phenols
—
1.10b
2.03a
Oxygenated sesquiterpenes
11.04a
5.93c
8.12b
Labdane type diterpenes
5.61a
2.22b
0.59c
Total
92.03
a
80.17
c
80.79
b
*Components are listed according to their elution on apolar column (HP-5). RI: retention indices relative to C8–C22  n-alkanes on the aHP-5 and bHP-Innowax columns; nd: not detected; GC/MS: identification based on comparison of mass spectra; Co-GC: identification based on retention time comparison to authentic compounds. Values (means of three replicates) in the same lines with different letters (a–c) are significantly different at P<0.05.The essential oil ofS. officinalis seeds showed a higher percentage of monoterpenes (56.59%) than sesquiterpenes (17.32%). Oxygenated derivatives were major among the monoterpenes (50.14%), while they represented only 5.93% of sesquiterpenes. Seeds essential oils were characterised by the predominance of α-thujone (14.77%), camphor (13.08%), and 1,8-cineole (6.66%). Viridiflorol (2.66%) and α-humulene (3.71%) were also detected in seeds essential oil of sage. Furthermore, other minor compounds were found, especially the labdane type diterpene 13-epi-manool.Essential oil compounds ofS. sclarea seeds were representing 80.79% of total essential oil components. The essential oil is predominated by monoterpenes accounting for 47.98%; their oxygenated derivatives (38.38%) prevailed on hydrocarbon ones (9.60%). The sesquiterpenes pool is less numerous (29.39%). In addition, one diterpene compound, 13-epi-manool, is detected at a level of 0.59% and some phenols (2.03%) such as thymol and carvacrol were produced in small amounts (0.1% and 1.93%, resp.). The oxygenated monoterpenes were characterised by linalool (24.25%), geraniol (2.79%), and their ester derivates (linalyl acetate (6.90%) and geranyl acetate (1.94%)). The sesquiterpenes characteristics of seeds were germacrene-D (5.88%), bicyclogermacrene (4.29%), and α-copaene (4.08%).


## 5. Discussion

As forS. verbenaca, the oil yield was higher than that offered by seeds originated from Tunisia [12]. Compared with leaves ofS. verbenaca from Tunisia [14], seeds herein studied appeared as moderately rich in volatile oil. Recovered essential oil fromS. sclarea seeds appeared to be near to literature data which showed that inflorescences oil yield of a cultivated strain developed in India [23]. InS. officinalis, the fruits of sage cultivated in Tunisia are distinguished by oil yields of 0.39% [24]. Thereby seeds appeared as oil-moderate organs contrary to the different other parts of the species owing to the genusSalvia its aromatic reputation amongst Lamiaceae family.As regards essential oil composition ofS. verbenaca, it is worthy to note that tricyclene and camphor are also common to the seeds sample from Tunisia [12] so we may suggest that these two compounds are significant markers of essential oil compounds ofS. verbenaca seeds whatever the sample origin is. Interestingly, the essential oil of the studied seeds could be employed as antimicrobial agent since their high percentage of camphor is associated with an efficient antimicrobial activity according to Magiatis et al. [25] and Bougatsos et al. [26].InS. officinalis, the predominance of α-thujone, camphor, and 1,8-cineole endowed to the essential oil an antimicrobial activity [27]. Viridiflorol and α-humulene identified in severalS. officinalis essential oils showed antiacetylcholinesterase activity used in the treatment of Alzheimer’s disease [28], antifungal property [29], and cytotoxic activity against some tumor cell lines [30]. Furthermore, the labdane type diterpene 13-epi-manool displaysin vitro a cytotoxic activity against human leukemic cell lines [31]. We noted that seeds had a similar qualitative composition to aerial parts with the predominance of α-thujone, β-thujone, camphor, and 1,8-cineole. These monoterpenes are taken as significant parameter to differentiateS. officinalis from other species [32]. Their amount is lower than that in leaves but matches the ranges of the standard ISO 9909 for official sage oil except for the toxic ketone α-thujone which had a lower amount (14.77%). These findings promote the use of the seeds essential oil in food industry.Similar to the studiedS. sclarea essential oil, the sesquiterpenes were mainly composed of germacrene-D, α-copaene, and bicyclogermacrene in plant inflorescence according to Carrubba et al. [11] and Lorenzo et al. [33]. In our sample, the essential oil ofS. sclarea flowering shoots raised in experimental plots in India was characterised by linalool (36.6–41.9%) and linalyl acetate (13.2–19.2%) as main compounds [23]. The wild-growingS. sclarea collected at flowering stage from central Greece [7] and Spain [6] showed a close similar composition regarding linalool and linalyl acetate (30.43%, 32.97% and 19.75%, 16.85%, resp.). Generally,S. sclarea essential oil is extracted from flowering shoots which are found to be rich in linalool and linalyl acetate. According to Carrubba et al. [11] high amounts of linalool and linalyl acetate are typical of good quality oil suitable for flavouring purposes. Furthermore, linalool plays a major role as anti-inflammatory suggesting that linalool-producing species are potentially anti-inflammatory agents [34]. Moreover, linalool has an ecological role since it constitutes one of the common components of floral scent that can attract a large variety of insects that convey pollen [35]. The present study showed that essential oil derived from seeds had a similar composition to the flowering parts. Thus, seeds seemed to display the same enzymatic patterns of the essential oil biosynthesis as the flowers, while the essential oil composition ofS. sclarea vegetative organs displayed different qualitative trends from reproductive parts.


## 6. Conclusions

Overall, it emerges that tricyclene and camphor were biochemical markers of the essential oil ofS. verbenacaseeds. Being rich in camphor, seeds could be used as antimicrobial agent. Another point that should be highlighted is thatS. officinalisseeds had the same α-thujone chemotype as leaves, whereas these two organs showed some quantitative differences leading to the safe use of seeds essential oil in food industry. From a qualitative standpoint, seeds ofS. sclarea seemed to have the same enzymatic trend as flowers characterized by the prevalence of linalool. It is noteworthy to mention that linalool-producing seeds asS. sclarea were suitable for flavouring purposes and constitute potential anti-inflammatory agents.



---

*Source: 838162-2014-08-27.xml*