2.1. Chemical Composition of SEO and TEO
In Table 1
and Table 2
are presented the results regarding the chemical composition of analysed EOs and their liniar retention indices (LRI).
In SEO, there have been identified 14 major compounds, in a concentration over 0.2%. The main compounds identified in this essential oil, at a level over 10%, were: caryophyllene (25.364%), camphene (14.139%), eucalyptol (13.902%), and β
-pinene (11.230%), representing 64.635% from all analysed components, Table 1
. Also, other compounds as thymol (8.073%), camphor (4.028%), and valancene (5.525%) have been found in a proportion over 4%. The analysed compounds are divided in monoterpene hydrocarbonates (37.387%), sesquiterpene hydrocarbonates (25.364%), and monoterpene oxigenated (34.874%).
Our previous study has identified the presence of camphor (20.4%), eucalyptol (11.7%), camphene (11.5%), α
-pinene (9.5%) as major compounds of the SEO that has been isolated from sage originating from western Romania [26
]. Camphor has been reported as the main compound in different other studies [31
], while other authors have identified α
-thujone as main chemical compound [32
]. The major similarity between the studies around the world regarding the SEO composition is represented by the fact that α-thujone was identified as the major compound, followed by camphor in a percentage of 20–25% of the total amount of chemical compounds.
In TEO, the γ
-terpinene and thymol were identified as the major compound in proportion of 68.415% and 24.721% respectively. These results are in agreement with those previously published by other authors, who reported that the major component of thyme essential oil is thymol, a monoterpene phenol derivative of cymene [32
]. Rus et al. [35
] have reported that o
-cymene and γ
-terpinene are the main chemotypes of TEO. Regarding the type of compounds, monoterpene hydrocarbonates (MH) represent 68.415%, monoterpene oxigenated (MO) 25.299%, and sesquiterpene hydrocarbonates (SH) 6.063%.
The content of volatile compounds and the dominant chemotype varies with botanical specie, geographical origin, and the harvest time. Therefore, the study carried out by Imelouane et al. [36
] have revealed camphor as the major compound (39.390%) of Thymus vulgaris
L. essential oil cultivated in Morocco, while Hudaib et al. [33
] have identified carvacrol, as the main chemotype of this essential oil. In another study conducted by Schmidt et al. [32
], linalool has been found as the major compound of Thymus vulgaris
L. essential oil (72.5%).
2.2. Cytotoxic Effect
Among the two volatile oils, SEO was more active in terms of inhibition of proliferation for the two tested melanoma cell lines. The inhibitory activity was linear with the concentration and quite similar for A375 human melanoma and B164A5 mouse melanoma cell lines. The lowest tested concentration of SEO (50 μg/mL) caused an inhibition ratio of 34% for the murine melanoma cell line and 39% for the human melanoma cell line, while a tested concentration of 100 µg/mL SEO lead to an inhibition ratio of 50.5% for the murine melanoma cell line and 47.5% for the human melanoma cell line. TEO at a tested concentration of 50 µg/mL induced an inhibition ratio of 19.5% for B164A5 cell line and 17.5% for A375 cell line, while a concentration of 100 µg/mL resulted in an inhibition ratio of 30% for B164A5 cell line and 27% for A375 cell line, Figure 1
. Regarding the essential oils mixture (EOsM), the use of a concentration of 50 µg/mL conducted to a percentage of inhibition of 30% for B164A5 cell and 26.5% for A375 cells. The antiproliferative effect increases with the concentration of EOs.
In vitro antiproliferative activity against M14 human melanoma cells was reported for the essential oil obtained from other species of sage originating from Lebanon namely Salvia bracteata
Banks & Sol and Salvia rubifolia
]. In a comprehensive study, Russo et al. [15
] have analyzed the chemical composition, and the growth-inhibitory and pro-apoptotic properties against A375, M14, and A2058 human melanoma cell lines of the SEO grown in eighteen different environmental conditions. The species showed in a different manner antiproliferative and pro-apoptotic properties on the selected melanoma cell lines, thus, demonstrating that the environmental and pedoclimatic conditions have a vital role on the chemical composition of volatile oil, and also, on the biological activity. α
- and β
-thujone isomers where assigned as responsible for the anti-melanoma activity, acting synergic with the other compounds present in the volatile oil [15
It has been proven that the EO of Salvia libanotica
showed in vivo chemoprevetive properties against skin cancer, experimentally induced with 7.12 dimethylbenz[a
]anthracene (DMBA) and 12-0-tetradecanoylphorbol-13-acetate (TPA) [37
The study conducted by Privitera et al. [38
] investigated the antiproliferative activity of SEO at a level of 200 µg∙mL−1
for 72 h of incubation on two human lung cancer cell lines (A549 and NCI-H226), and significantly inhibition has been detected [38
Together with the volatile oil obtained from other aromatic plants, the antiproliferative activity of the volatile oil of Salvia triloba
L. was studied for MCF7 human breast adenocarcinoma cells. Interestingly, the ethanol extracts presented antiproliferative capacity but the aqueous extracts and the pure volatile oil, not [13
]. In a broad study, analyzing the effect of the SEO, Loizzo et al. [39
], have concluded that the cytotoxic effects were remarked for C32 amelanotic melanoma cell line, with an IC50 of 367 μg/mL and for ACHN renal adenocarcinoma human cell lines, with an IC50 of 108 μg∙mL−1
. On the other hand, no effects were reported for LNCaP prostate carcinoma cell lines and MCF-7 human breast cancer cells. Evaluating the anticancer effects of Salvia officinalis
L. volatile oil against UMSCC1, the squamous human cell carcinoma cell line of the oral cavity have described a dual effect, including both stimulation and inhibition, depending on the concentration. IC50 value has been reported to be 135 μg∙mL−1
]. Itani et al. [41
] have noticed that three major components of Salvia libanotica
EO, namely linalyl acetate, terpeniol, and camphor caused growth inhibition and apoptosis for human colon cancer HCT116 cells both p53+/+
The volatile oil of thymus endemic species from Algeria and Morocco was found to show cytotoxic properties against A375 human melanoma cell line with an IC50 46.95 μg·mL−1
]. A recent study depicted the antiproliferative activity against THP-1 human monocytic cell line of the volatile oil obtained from different species of thymus originating from Portugal [43
]. The findings were supported by the group of Tefiani et al. [44
] that concluded that Thymus munbyanus
volatile oil collected from Algeria present remarkable antiproliferative effects against THP-1 human acute monocytic leukemia cell line.
The essential oils of two thymus species, namely Thymus linearis
Benths., and Thymus serpyllum
L. from the Pakistan flora, have been screened for antiproliferative effects against two human cancer (MCF-7-breast and LNCaP-prostate carcinoma) and one fibroblast (NIH-3T3) cell lines. Both volatile oils showed antiproliferative potential on the above mentioned cell lines, but the effectiveness of the volatile oil obtained from Thymus linearis
Benths. has been significantly increased with IC50 of 80.7 μg·mL−1
, 70.3 μg·mL−1
and 100.2 μg·mL−1
]. A recent study has shown the cytotoxic effect of Thymus caramanicus
Jalas, a common species in Iran, against KB human oral epidermoid carcinoma [46
2.3. Antifungal Activity
In Table 3
, the results regarding the antifungal activity of SEO and TEO are shown on Fusarium graminearum
mycelium growth area (MGA). At a closer look, it can be noted that the highest radial grow increase of mycelium was recorded in sample treated with SEO at level of 0.06%, where the MGA value was about 15.9 cm2
. By applying of SEO and TEO mixture at a level of 0.06% (in the same proportion) was obtained only 0.1 cm2
value of MGA. This finding reveals the synergistic effect of SEO and TEO. Similarly, the previous studies on this topic have shown that the antifungal effect is not only caused by a major compound as by the synergy of the other compounds found in smaller amounts [47
With respect to the antifungal potential of TEO, our results revealed a total inhibition of Fusarium
mycelium growth at all investigated concentrations. Thus, the mycelia radial growth (RG) in sample with TEO was similar to thiophanate methyl (as negative control), the MGA values being null (in Table 3
Although by applying the EOsM at 0.06% an increase of mycelium was recorded, the differences are not statistically significant (p
> 0.05) versus negative control. The antifungal effect of sage oil has been highlighted on other fungal species. Mahmoudi et al. [49
] have investigated the effect of SEO on Alternaria alternata
, and reported that a level of 5 mg/L was the minimum inhibitory concentration against the fungus growth. The same effect has been shown for Botritis cinerea
]. Additionally, it has been proved that the SEO showed antifungal activity against dermatophyte strains [52
The antifungal potential of TEO has been demonstrated also on other types of fungi. Thus, the study performed by Tantaoui-Elaraki et al. [53
] investigated the effect of TEO on Aspergillus parasiticus
in vitro conditions and reported that the minimum inhibitory concentration for this type of fungi was 0.1%. Also, it has been proved that TEO has shown a strong antifungal activity on Verticillium dahliae
sp., and Aspergillius
]. Among Thymus
, and Eucalyptus
, essential oils it has been found that TEO showed the lowest inhibitory concentration against the fungal growth [55
2.4. Allelopatic Potential
The results regarding the herbicidal effect of the investigated EOs compared to positive control as synthetic herbicide Dual Gold (960 g L−1
s-metolachlor), (DG) are presented in the Figure 2
. The DG completely inhibits the weeds germination, while the tomato seeds are inhibited in percentage of 76.8% and wheat seeds in proportion of 47.2%.
Our results have proven that SEO showed herbicidal effect on Amaranthus retroflexus L. (ARET), even when it was used at low concentrations. Thus, when SEO was applied at a level of 0.3%, the inhibition of germination was 92.1%, while by increasing the level of SEO to 0.6% or 1.0%, total inhibition of germination of ARET seeds occurred. Controlling the germination of Chenopodium album L. (CALB) weeds by using Dual Gold is 100% effective at recommended dose of 1 L per hectare. The same value of germination inhibition rate (GI) of CALB weed is also achieved by spraying the seeds with SEO at a level of 1.0%. The herbicidal capacity of SEO decreases with the dose. Thus, by applying SEO at a level of 0.6% resulted of 55.5% value of GI, while a dose of 0.3% SEO led to 22.2% value GI.
The herbicidal effect of SEO on Echinochloa crus-galli L. seeds (EGAL) has reached a maximum value of GI about 75% by applying SEO at a level of 0.6% and 1.0%, while a level of 0.3% SEO induced the lowest herbicidal effect of 69.1%.
The study of the allelopathic effect of EOs on wheat and tomato seeds shows that the SEO, applied at a level of 0.6% resulted in a GI value of 88.4% while a level of 1.0% has the ability to inhibit the germination in a percentage of 100% for tomato seeds and 80.5% for wheat seeds.
Using SEO al a level of 0.3% it was noted a decrease in the germination inhibition of tomato seeds up to 14.8%, respectively up to 8.3% in wheat seeds, as compared to the synthetic herbicide that showed a better ability to inhibit the seeds germination (76.8% for tomatoes and 47.2% for wheat). Thus, there are no significant differences versus DG as positive control by using SEO as an herbicidal agent for ARET, CALB seeds (except 0.6% SEO when the statistical differences were significant, p < 0.05) and EGAL (except 1.0% SEO when the statistical differences were significant, p < 0.05). SEO applied at a level of 1.0% induced significant difference (p < 0.05) versus positive control in GI of tomato seeds, while no significant differences versus positive control were recorded in the GI of wheat seeds.
By applying TEO at different doses, only for a level of 0.3% there were recorded significant differences (p < 0.05) in the GI of ARET, EGAL, and tomatoes versus DG as a positive control.
As regards the essential oils mixtures (EOsM), when was applying at a level of 0.3% in the same proportion, significant differences were recorded in the GI of ARET versus positive control. Also, the using of 0.6% EOsM induced significant differences in the GI value of EGAL versus the positive control.
All of the investigated levels of EOsM induced no significant differences in GI of wheat seeds versus positive control, while significant differences were noted in the GI of tomato seeds versus positive control.
The ability of SEO at a level of 0.3% to inhibit the germination of weed seeds and to slightly affect the germination of wheat and tomato seeds could recommend the use of this essential oil in plant protection.
Our results have proven that the TEO shows better herbicidal potential than SEO. Thus, at TEO applied at a level of 0.6% and 1.0% has completely inhibited the germination of CALB and EGAL seeds and in a proportion of 60.6% in the case of ARET seeds.
By decreasing the level of TEO at 0.3% do not record a great inhibition of the seeds germination of ARET (6.6%) and CALB (10%). Unfortunately, TEO showed an inhibition effect on organic tomato and wheat seeds, at over 80%, even used at low doses.
The synergistic effect of the investigated oils resulted in increasing the allelopathic potential of their mixture and in a total inhibition of germination on all types of weeds, when EOsM was applied at a level at least 0.6%. It is worth noting that the use of the mixture does not inhibit the wheat seeds germination, thus recommending the blending of the two EOs in equal proportion, as bio-effective herbicides in the control of weed grain crops.
When compared to synthetic herbicide DG, the investigated EOs mixtures exert an increased efficiency without having a negative effect on the wheat germination. However, the synergistic effect of the two EOs, even applied at a low level, leads to a complete inhibition of tomato seeds germination.
The inhibitory capacity of EOs applied both individually and as a mixture, can be strongly influenced by their chemical composition and also by the generated synergism. It has been reported that the terpenoids, particularly the sesquiterpene, even at a low level, exhibited specific structure-activity relationship, which can generate phytotoxicity [56
The previous studies have highlighted the bio-herbicidal effects of EOs belonging to Lamiaceae family (oregano and rosemary) on weeds control and germination growth of bread wheat cultivars [31
]. The results have shown that 2–4 μL of EOs exhibits an inhibition effect on Sinapis arvensis
L., but not against Avena sterilis
L. seeds. Moreover, these seeds have survived at all doses of oregano and rosemary EOs. Other studies [57
] revealed the inhibition potential of peppermint, cinnamon, and lavender oils against redroot pigweed, while ryegrass and wild mustard seeds germination. When compared with the control, peppermint oil has been the more effective in inhibiting ryegrass seed germination. Study regarding the effect of eucalyptus, camphor, and lemongrass EOs on seed germination and seedling growth of Parthenium hysterophorus
shown that a higher level than 12 mL/L induced a total inhibition of weed germination [56
Our results revealed significant differences in the inhibitory effect of the investigated EOs on germination capacity of wheat and tomato versus weed species. These findings could be assigned to the different rates to metabolize certain monoterpenes [31
]. Thus, the volatile compounds may also be responsible for the inhibition of seed germination [58
]. Wheat and tomato seeds were less affected than the weed seeds species, which highlights that the EOs, applied in a proper dose could be recommended as a bio herbicide for weed control.