Chemical Composition and Antigerminative Activity of the Essential Oils from Five Salvia Species

The chemical composition of the essential oils of Salvia africana L., Salvia elegans Vahl, Salvia greggii A. Gray, Salvia mellifera Green and Salvia munzii Epling, cultivated in Eboli (Salerno, Southern Italy), was studied by means of GC and GC-MS analyses. In all, 88 compounds were identified, 54 for S. africana, accounting for 95.4% of the total oil, 55 for S. elegans (92.9%), 50 for S. greggii (96.9%), 54 for S. mellifera (90.4%) and 47 for S. munzii (97.5%), respectively. In S. africana, the amount of monoterpenoids and sesquiterpenoids is very similar. For other species, the monoterpenoid percentage is greater than the amount of sesquiterpenoids. The oils of S. elegans, S. greggii and S. munzii were active inhibitors of germination and radical elongation of Raphanus sativus L. and Lepidium sativum L.


Introduction
Allelopathy is an expression of the general chemical interaction among plants: a large number of plants possess both inhibitory and stimulatory effects on the growth of neighbouring or successional plants by releasing chemicals into the soil [1][2][3]. The study of plant compounds, which inhibit or OPEN ACCESS stimulate the germination and the development of other species, is important for understanding the mechanisms of the ecological interaction. Our research group is studying the possible allelopathic effects of medicinal and aromatic plants [4][5][6][7] that, being rich in active principles, are considered an important source of potential allelochemicals.
The genus Salvia (Lamiaceae: subfamily Nepetoideae, tribe Mentheae) is a cosmopolitan assemblage of nearly 1,000 species showing a remarkable diversity in growth forms, secondary compounds, floral morphology and pollination biology. "Salvia phenomenon" is one of the best known and well-studied examples of allelopathy [8]: Muller and co-workers showed the potent potential allelopathic of Salvia leucophylla Greene and S. apiana Jeps. [8][9][10][11][12].
Salvia africana L. is an aromatic, hardy shrub up to 2 m in height, originating from Africa. There are no literature data about the essential oil of Salvia africana L. Salvia elegans Vahl is a perennial shrub native to Mexico, commonly known as "pineapple sage" and "pineapple-scented sage" in English, and "mirto", "flor del cerro", "limoncillo" and "perritos rojos", in Spanish. The volatiles of pineapple-scented sage were analyzed for the first time by Makino and coworkers [13]. S. elegans is widely used in Mexican traditional medicine for alleviate Central Nervous System ailments [14]; Herrera-Ruiz and co-workers reported this species as a possible source for isolating new anxiolytic and antidepressant substances [15]. Moreover, Wake and coworkers studied this species for its cholinergic activity [16]. Salvia greggii A. Gray, "autumn sage" or "autumn salvia", a biennial plant originating from both Mexico and the Texas, is a semi-woody species that has a showy display of brilliant red, pink, white or orange flowers from spring until the first frost in fall [17]. Only few phytochemical reports are available about S. greggii, in particular concerning its diterpenoid compounds [18][19][20]. Salvia mellifera Greene grows abundantly in California at heights below 2,000 feet and it undergoes various hybridizations with other species of the same genus [21]. S. mellifera is a dominant species in much of the California coastal scrub sage and bordering chaparral. Several studies reported the chemical composition of this species [22][23][24] and the biological activity of its volatile terpenes [8]. In northernmost Baja California as the coastal sage scrub becomes increasingly xeric, S. mellifera is replaced by S. munzii [22]. The chemical composition of the essential oil of S. munzii was studied before [22], showing the presence of camphor, 1,8-cineole and limonene, as main constituents.
In the S. mellifera oil, the monoterpene fraction amounted to 76.1%, while the sesquiterpene one was 14.2%. Also in this case, the monoterpene fraction was mainly represented by oxygenated compounds (57.0%), with great prevalence of 1,8-cineole (39.8%) and camphor (12.2%). α-Pinene (9.2%) was the major component of non-oxygenated monoterpenes. Neisess and coworkers [22] reported camphor as one of the most abundant compounds in S. mellifera and S. munzii oils; our data confirmed their results.
In S. munzii, the monoterpene fraction amounted to 80.3% of the total oil, while sesquiterpenes represented only 15.6%: the main compounds are cis-thujone (33.3%) and camphor (27.2%), with δcadinene (8.9%) and geraniol (4%) in appreciable amounts. In literature, Neisses and coworkers [22] reported the essential oil composition of this species and they showed that camphor is one of the major compound of the oil: once more, our data confirmed their results.

Biological activity
The five essential oils were evaluated for their phytotoxic activity against germination (Table 2) and initial radical elongation (Table 3) of radish (Raphanus sativus) and garden cress (Lepidium sativum), two species usually utilized in biological assays [7]. The oils affected the germination and the radical elongation of two seeds in a distinct way. The germination of radish appeared sensitive to Salvia greggii oil, at the highest dose (1.25 μg/mL) used. The germination of garden cress was completely inhibited by S. elegans, S. greggii and S. munzii oils, at the highest doses (1.25 μg/mL, 0.625 μg/mL) used. The essential oil of S. elegans, at the almost all doses tested, inhibited significantly the radical elongation of both radish and garden cress. Also S. greggii and S. munzii oils inhibited, in a significative way, the radical elongation as of radish as of garden cress; on the other hand, S. mellifera oil inhibited, in a significative way, the radical elongation of radish but not of garden cress, while S. africana oil was inactive towards both seeds. 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 9.3 ± 0.6 0.06 μg/mL 9.7 ± 0.6 8.3 ± 1.5 9.3 ± 1.1 8.7 ± 0.6 9.7 ± 0.6 0.125 μg/mL 9.7 ± 0.6 8.7 ± 1.5 8.0 ± 1.7 8.7 ± 0.6 9.0 ± 1.0 0.25 μg/mL 10 ± 0 6.0 ± 1.0* 7.0 ± 1.0 7.0 ± 1.0 10 ± 0 0.625 μg/mL 10 ± 0 0 ± 0 1 ± 0 6.0 ± 1.0 * 0 ± 0 1.25 μg/mL 9.0 ± 1.0 0 ± 0 0 ± 0 6.3 ± 0.6 0 ± 0 Note:p < 0.05 vs. control.  The difference in biological activity of the oils could be attributed to their different chemical composition: in S. africana oil, there is a minor amount of oxygenated terpenoids, reported as germination and seedling growth inhibitors [27], in comparison with the other four oils: in fact, this species of Salvia is the less active. On the other hand, the oils of S. elegans, S. greggii and S. munzii, that have a similar chemical composition, with cis-thujone, camphor and 1,8-cineole as main components, possess a comparable antigerminative activity. These compounds are known as potent inhibitors of seed germination [8]. Moreover, Pinto and coworkers [28] showed that a Salvia officinalis oil, containing 10.4% of cis-thujone and 20.5% of camphor, was very active against Candida sp., dermatophytes and other filamentous fungi and showed fungicidal activity: cis-thujone and camphor are both present in our most active oils.
Our data agree with the literature on inhibitory activity exerted by essential oils of Salvia species on seed germination and radical elongation and, in general, on vegetation. Muller [12] reports a dramatic example of zones free of annual herbs, influenced by terpenoids, in the areas surrounding patches of Salvia leucophylla. Several authors [29][30] studied the mechanism of monoterpene volatilization in S. mellifera. Volatile monoterpenoids, emanating from leaves of this species are responsible for anatomical and physiological changes occurring in herb seedlings which were exposed to vapours [31]. Camphor and 1,8-cineole, the main components of the oil of Salvia leucophylla, are potent inhibitors of oxygen uptake by mitochondrial suspensions [12]. Moreover, we reported recently the phytotoxic activity of other two species of Salvia [26].

Isolation of the volatile components
Fifteen grams of each air-dried sample of Salvia species were ground in a Waring blender and then subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia [39]. The oils were solubilised in n-hexane, filtered over anhydrous sodium sulphate and stored under N 2 at +4 °C in the dark until tested and analyzed. The dry materials gave yellowreddish oils in a yield of 0.37% (v/w) for S. africana, of 0.55 (v/w) for S. elegans, of 0.70% (v/w) for S. greggii, 0.68% (v/w) for S. mellifera and 0.80% (v/w) for S. munzii.

Gas chromatography
Analytical gas chromatography was carried out on a Perkin-Elmer Sigma-115 gas chromatograph equipped with a FID and a data handling processor. The separation was achieved using a HP-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Column temperature: 40 °C, with 5 min initial hold, and then to 270 °C at 2 °C/min, 270 °C (20 min); injection mode splitless (1 μL of a 1:1,000 n-pentane solution). Injector and detector temperatures were 250 °C and 290 °C, respectively. Analysis was also run by using a fused silica HP Innowax polyethylenglycol capillary column (50 m × 0.20 mm i.d., 0.25 μm film thickness). In both cases, helium was used as carrier gas (1.0 mL/min).

Gas chromatography-Mass spectrometry
Analysis was performed on an Agilent 6850 Ser. II apparatus, fitted with a fused silica DB-5 capillary column (30 m × 0.25 mm i.d., 0.33 μm film thickness), coupled to an Agilent Mass Selective Detector MSD 5973; ionization energy voltage 70 eV; electron multiplier voltage energy 2,000 V. Mass spectra were scanned in the range 40-500 amu, scan time 5 scans/s. Gas chromatographic conditions were as reported in the previous paragraph; transfer line temperature, 295 °C.

Identification of components
Most constituents were identified by gas chromatography by comparison of their Kovats retention indices (Ri) with either those of the literature [40,41] or with those of authentic compounds available in our laboratories. The Kovats retention indices were determined in relation to a homologous series of n-alkanes (C 8 -C 28 ) under the same operating conditions. Further identification was made by comparison of their mass spectra on both columns with either those stored in NIST 02 and Wiley 275 libraries or with mass spectra from the literature [40,42] and a home made library. Components relative concentrations were obtained by peak area normalization. No response factors were calculated.

Biological assay
A bioassay based on germination and subsequent radical growth was used to study the phytotoxic effects of the essential oils of S. africana, S. elegans, S. greggii, S. mellifera and S. munzii on seeds of Raphanus sativus L. cv. "Saxa" (radish), and Lepidium sativum L. (garden cress). The seeds were purchased from Blumen srl, Piacenza, Italy. The seeds were surface sterilized in 95% ethanol for 15 s and sown in Petri dishes (Ø = 90 mm), containing five layers of Whatman filter paper, impregnated with distilled water (7 mL, control) or tested solution of the essential oil (7 mL), at the different assayed doses. The germination conditions were 20 ± 1 °C, with natural photoperiod. The essential oils, in water-acetone mixture (99.5:0.5), were assayed at the doses of 1.25, 0.625, 0.25, 0.125 and 0.062 μg/mL. Controls performed with water-acetone mixture alone showed no appreciable differences in comparison with controls in water alone. Seed germination was observed directly in Petri dishes, each 24 h. Seed was considered germinated when the protrusion of the radical became evident [43]. After 120 h (on the fifth day), the effects on radical elongation were measured in cm. Each determination was repeated three times, using Petri dishes containing 10 seeds each. Data are expressed as the mean ± SD of both germination and radical elongation. The Student's t test of independence was applied [44].

Conclusions
Aromatic plants are regarded as a primary source of potential allelochemicals and interact with their environment. Muller and coworkers demonstrated [8][9][10][11][12] that Salvia species produce volatile growth inhibitors, particularly oxygenated monoterpenoids. These findings were subsequently confirmed by other papers [13,29]. Our in vitro experiments on the essential oils from Salvia species on germination and initial radical elongation of radish and garden cress, show that the essential oils of S. elegans and S. munzii were the most active inhibitors, whereas S. africana oil didn't show such activity. The phytotoxic activity of S. elegans and S. munzii was probably due to the presence of a substantial amount of oxygenated terpenoids, in particular of cis-thujone, 1,8-cineole and camphor. Our in vitro studies can contribute to explain the importance of volatile compounds as chemical mediators in biochemical interactions among higher plants and could suggest models for lead compounds in the development of new pesticides [45].