4.1 : GC-MS analysis and E.O. composition. 4.1.1 : Essential Oils from

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4.1 : GC-MS analysis and E.O. composition.

Table 4.1: Main Constituents in EOs from Aerial Parts and Flowers of S. officinalis Relative Percentage %

Compound L.R.I. Aerial Parts Flowers

α-pinene 930 5,4 5,6 camphene 946 1,9 5,5 β-pinene 974 2,3 16,9 1,8-cineole 1033 7,1 18,5 α-thujone 1103 27,8 19,3 camphor 1129 16,7 3,1 β-caryophyllene 1419 4,5 5,1 α-humulene 1450 7,0 3,9

Both oils were characterized by the high levels of the oxygenated monoterpenes (60.2%% in the aerial parts and 47.5% in the flowers; see table..). The hydrocarbon monoterpenes were present in higher percentage in the flower (31.3%) respect to the aerial parts (14.2%). In both essential oils the most abundant compound was α-thujone (27.8% in the aerial parts and 19.3% in the flowers). Other main constituents present in the EO from the aerial parts were the oxygenated monoterpenes: camphor (16.7%) and 1,8-cineole (7,1%), while the main sesquiterpenes were β-caryophyllene (4.5%) and its isomer α-humulene (7.0%). In the flowers EO profile together with α-thujone, the percentages of β-pinene (16.9%) and 1,8-cineole (16.9%) were up to 15%.

4.1.2 : Essential Oil from Salvia dolomitica.

Table 4.2: Main Constituents in Salvia dolomitica Essential Oil

Compound Rt (min) L.R.I. Relative Percentages

α-pinene 5,29 939 6,4

3-carene 7,59 1011 5,5

limonene 8,24 1031 11,0

1,8-cineole (eucaliptol) 8,33 1033 13,9

β-caryophyllene 24,94 1420 5,3

The oil was characterized by the high levels of the hydrocarbon monoterpenes (39.6%), oxygenated monoterpenes (20.9%) and hydrocarbon sesquiterpenes (25.3%) while the hydrocarbon sesquiterpenes were present in minor percentage (8.4%). The main constituents were limonene (11.0%), 1,8-cineole (13.9%) and α-pinene (6.4%).

128 4.1.3 : Essential Oil from Salvia patens.

Table 4.3: Main constituents in S. patens essential oil

Compound Rt (min) L.R.I. Relative Percentage %

β-pinene 6,54 978 7,2

α−gurjunene 24,23 1410 10,0

β-caryophyllene 24,77 1420 5,6

germacrene D 27,44 1485 10,4

bicyclogermacrene 28,04 1500 7,9

The oil was characterized by the high levels of the hydrocarbon monoterpenes (39.6%), oxygenated monoterpenes (20.9%) and hydrocarbon sesquiterpenes (25.3%) while the hydrocarbon sesquiterpenes were present in minor percentage (8.4%). The main constituents were limonene (11.0%), 1,8-cineole (13.9%) and α-pinene (6.4%).

4.1.4 : Essential Oil from Salvia somalensis. Table 4.4: Main constituents in S. somalensis essential oil

Compound L.R.I. Relative Percentage %

3-carene 1013 6,4 camphor 1154 12,8 bornyl acetate 1286 15,2 β-caryophyllene 1419 4,4 cis-γ-cadinene 1511 1,5 trans-γ-cadinene 1513 3,6

The EO were characterized by high percentages of monoterpenes derivatives (74.0%), in particular, hydrocarbon monoterpenes were 40.2% and hydrocarbon monoterpenes were 33.8%. The sesquiterpenes representing 24.7% of the total amount while the hydrocarbon compounds were absent. The main constituents founded were camphor (12.8%) and bornyl acetate (15.2%).

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4.2 : Antibacterial activity via Disc Diffusion Assay.

The essential oils tested displayed a certain activity against one or more bacteria, but the concentrations needed were very high in most cases. It probably cannot be possible to use them as new antibiotic agents.

Not all the oils tested showed an inhibitory effect on all the bacteria tested; for example

Salvia patens was not effective at all against Pseudomonas aeruginosa. The oils which presented an antibiotic activity showed a certain trend linking concentration and zone of inhibition: the higher the concentration, the wider is the diameter of the zone of inhibition. This meant that the procedure used for testing was good.

The standard deviation between the three replicates resulted acceptable, so that repeatability was assured.

The most active essential oil on Staphylococcus aureus was Salvia patens, especially at the highest concentration of 160 µg/µL. Under 80-40 µg/µL all oils usually lost their activity.

The most active essential oil on Staphylococcus epidermidis was Salvia officinalis flowers, active even at ppm concentrations (till 6.25 ppm). Comparing the µg/µL concentrations, Salvia officinalis flowers is still the most active oil even if it was tested with a slightly different method.

The most active essential oil on Enterococcus faecalis was Salvia patens, which had the most wide diameter of inhibition for 160 µg/µL, but Salvia dolomitica was more active at lower concentrations (till 2.5 µg/µL).

The most active essential oil on Escherichia coli was Salvia patens (till 2.5 µg/µL). The ppm concentrations which gave some apparent results were not to be considered, because in those experiments the negative control gave inhibition even larger as well. The most active essential oil on Pseudomonas aeruginosa was Salvia officinalis branches and leaves, at the highest concentration of 160 µg/µL. Salvia dolomitica showed a stronger activity than the others in the range of 80-40 µg/µL, and then it lost its activity as well as Salvia officinalis branches and leaves. Salvia patens was not active at all.

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4.3 : Suggestions for further works.

This study succeeded in its purpose to investigate the antimicrobial activity of the five essential oils tested and this was a great goal, but there were some points which can be improved to make the research more accurate:

1) Hydro-distillation is the most spread process to extract the plant material, but there are newer techniques which are able to reduce the time of extraction, reduce the heat used (and so the degradation of the chemicals) and produce a good- quality oil, such as the supercritical fluid extraction. Maybe it could be possible to compare the chemical composition and the bioactivity of both extracts.

2) The number of bacteria strains on which the oils were tested could be raised up to have a better knowledge of the antibacterial activity of Salvia species E.O. 3) Since in scientific literature there are a lot of articles about antifungal activity of

Salvia species E.O., it may be interesting to repeat these experiments with some fungal strain.

4) The main chemical component of each oil should be tested against these bacteria, in order to have the possibility to compare the activity of the whole oil with the activity of a pure molecule. For example germacrene-D, α-gurjunene and δ-gurjunene should be tested against Staphylococcus aureus, α-thujone, 1,8-cineole and β-pinene against Staphylococcus epidermidis, 1,8-1,8-cineole against

Enterococcus faecalis, germacrene-D, α-gurjunene and δ-gurjunene against

Escherichia coli, α-thujone and camphor against Pseudomonas aeruginosa. 5) The disc diffusion assay is a quite cheap technique and does not require a

particular equipment, but is a qualitative method and it has some limits already discussed in chapter 2. The research on these essential oils on these bacteria should be more complete if the experiments would be effectuated with a quantitative method (like micro-dilution). The MIC (minimum inhibitory concentration) and the MCC (minimum -cidal concentration) can give a better idea of the activity of the essential oil and its possible use as a drug.

Furthermore the micro-dilution method is usually combined with

spectrophotometric detection, so the concentration of the bacteria suspension would be more accurate than with a naked-eye comparison with the McFarland standard.