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Research Article

Supercritical Extraction of Salvia officinalis L.

V. Micic, Z. Lepojevic, M. Jotanoviae, G. Tadic and B. Pejovic
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The wild sage (Salvia officinalis L.) growing in municipality Trebinje, Bosnia and Herzegovina was extracted with supercritical carbon dioxide at flow rate 3.23•0-3 kg min-1, temperature 313 K under varying pressures in order to determine yields and compositions of extracts. Supercritical fluid extraction was carried out with a laboratory-scale high pressure extraction plant-HPEP (Nova-Swiss, Switzerland). With increasing pressure from 80 to 300 bar extraction yield increased. It was due to the fact that supercritical carbon dioxide density increases with increasing pressures. In the supercritical CO2 extracts the major components were phyllocladene (10.42-30.64%), γ-elemene (7.02-24.98%), isoborneole (6.80-11.29%), camphor (1.43-15.24%). Essential oils were isolated by hydro distillation from CO2 extracts. The major components were α-thujone (15.63-27.38%), camphor (16.03-23.45%), γ-elemene (7.46-15.52%). In continue of investigations, the kinetics of extraction where each point of the kinetic curves obtained with the new sample of drug in extractor at pressures 80, 100 and 200 bar were studied. With increasing pressure extract yield increased (1.40-4.17 g/100 g drug). Qualitative and quantitative analyses extracts and essential oils were done using GC/MS and GC/FID analyses. The composition of sage extracts and essential oils were different. Supercritical fluid extraction by CO2 allowed optimization of the experimental conditions for selection of the substances of interest.

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V. Micic, Z. Lepojevic, M. Jotanoviae, G. Tadic and B. Pejovic, 2011. Supercritical Extraction of Salvia officinalis L.. Journal of Applied Sciences, 11: 3630-3634.

DOI: 10.3923/jas.2011.3630.3634

Received: February 09, 2011; Accepted: April 20, 2011; Published: December 16, 2011


The past few years have seen a growing interest in natural foods, with increased demand for non-synthetic, natural antioxidants. The use of synthetic antioxidants in the food industry is severely restricted by low as to both application and level of use. Valued traditionally as a spice, sage is now being studied because of its antioxidant properties (Bozin et al., 2007; Ali, 2011).

Salvia officinalis L. is valuable medicinal plant which is used widely in traditional medicine. This plant is very rich in biologically active compounds and many studied have indicated their increasing practical importance (Velickovic et al., 2007). The presence of flavonoids in the sage has already been confirmed by Lu and Foo (2002) and Valant-Vetschera et al. (2003). They have therapeutic effects because of their inhibitory effects on certain enzymes and antioxidant activity. They have been shown to possess antibacterial (Harborne, 1986), antifungal (Weidenbornen and Jha, 1994), antiviral and anti-inflammatory activities (Abad et al., 1997). Their ant allergic, antioxidant and anti- mutagen activities (Yanishlieva et al., 1999; Hossain and Ismail, 2011) also been proven.

The demand of the food industry for natural antioxidant prepared with safe solvents has directed attention to more efficient extraction methods, such as Supercritical Fluid Extraction (SFE). Now, the conventional methods such as hydro-distillation and solvent extraction are unsatisfactory. The distillation procedure allows only the separation of volatile compounds (essential oils) which to a greater or lesser extent, are transformed under the influence of the elevated temperature (Koukos et al., 2000). On the other hand, extraction with organic solvents can hardly render an extract free of traces of the organic solvent which are undesirable for either organoleptic and/or health reasons. Besides, organic solvents are insufficiently selective, so that, in addition to the active substances, they also dissolve some concomitant compounds (Damjanovic et al., 2005). An alternative extraction technique with better efficiency and selectivity is highly require, in order to eliminate solvents, avoid the degradation or loss of sensitive and thermo-labile substances and to decrease the high energy and manpower input of conventional processes (Sovova, 2005; Stampar et al., 2006; Bendahou et al., 2007). To improve efficiency and selectivity of the extraction, alternative extraction techniques as supercritical extraction started to be developed.

The broad interest in supercritical CO2 extraction of essential oils is proved by large number of scientific literature published on this argument. These studies were undertaken in view of a possible industrial application of the process.

Supercritical extraction is not widely used yet but as new technologies are coming there are more and more viewpoints that could justify it, as high purity, residual solvent content and environment protection (Zekovic et al., 2001). The use of supercritical fluids (SCF) as reaction or separation media offers the opportunity to replace conventional organic solvents and also to optimize and potentially control the effects that solvent properties can have on selectivity.

Carbon dioxide is most widely used in Supercritical Fluid Extraction (SFE) because it is simple to use, inexpensive, non-flammable, nontoxic, chemically stable, shows great affinity to volatile (lipophilic) compounds and can be easily and completely removed from any extract (Reverchon and Senatore, 1992; Cheng et al., 2000; Adeib et al., 2010). By changing pressure and/or temperature above critical point of carbon dioxide (Tc = 31.3°C, Pc = 72.8 bar,) a pronounced change in the density and dielectric constant, i.e., solvent power of supercritical carbon dioxide can be achieved (Vargaftik, 1975; Ried et al., 1987). The balance between solvent power and selectivity of a supercritical solvent is perhaps the most important aspect to be optimized. Higher densities induce higher solvent power, however, solvent selectivity towards compounds characterized by similar polarities and different molecular weights decrease with this increase in solvent power. Therefore, supercritical CO2 can show high selectivity compared with liquid CO2 since its density varies from about 0.2 to 0.9 g cm-3 for many SFE conditions (temperatures from 40 to 60°C, pressures from 80 to 300 bar) (Vukalovich and Altunin, 1968). In processes performed at high CO2 densities the lower process selectivity associated with the higher extraction yield can result in the simultaneous extraction of several compound families and the co-extraction of compounds that do not contribute to fragrance formation. Since the odoriferous compounds, such as terpens, oxygenated terpens, sesquiterpenes and oxygenated sesquiterpenes are readily soluble in supercritical CO2, the extraction of essential oils at CO2 high densities is neither necessary, nor desirable (Reverchon et al., 1995).

The special properties of supercritical fluids bring certain advantages to chemical separation processes. Several applications have been fully developed and commercialized (Takeuchi et al., 2008; Wenqiang et al., 2007). The biggest application is the decoffeinization of tea and coffee. A process for removal of caffeine from coffee using supercritical carbon dioxide was patented in the United States in 1974 and a commercial plant went on stream in the FRG in 1978 (McHugh and Krukonis, 1994). Other important areas are the extraction of essential oils and aroma materials from spices. Brewery industry uses SFE for the extraction of hop (Zoran et al., 2007).

The influence of CO2 density and extraction time on the essential oil composition was studied.


Plant material: Sage leaves (Salvia officinalis L.) air dried in the shade were supplied in Berkovići, near municipality Trebinje. Leaves of growing wild sage were collected manually from the same collection site in the Berkovići region in 2008.

Chemicals: Commercial carbon dioxide (99% purity, Tehno-gas, Novi Sad, Serbia) as the extracting agent was used. All other chemicals were of analytical reagent grade.

Chromatographic procedures: GC analyses were performed using GC 5890 Series II (Hewlett-Packard, Palo, Alto, Calif, USA) gas chromatograph equipped with a FID and a DB-5 capillary column (30 mx0.25 mm, film thickness 0.25 μm). The hydrogen was used as a carrier gas with flow rate 1 mL min-1. Injector and detector temperature were 244 and 285°C, respectively. Oven temperature was programmed at 40°C for 2 min and then increased linearly to 285°C at a rate of 4.3°C min-1. A split flow of 1mL min-1 was used. The constituents of essential oil and extracts were identified by comparing their retention times with those of available standards and their mass was calculated from a predetermined peak area response factor.

The GC-MS analyses were carried on a capillary GC (Varian, model 3400) interfaced with an ion-trap detector (Finnegan Mat, model BE 8230) with the same column and same characteristics as the one used in GC. The samples, previously dissolved in chloroform : methanol (3:1), were injected (1 μL) in split mode with split ratio of 1:99 and the flow rate of carrier gas (hydrogen) was 1 mL min-1. MS conditions were: ionization voltage, 70 eV scanning interval 1.0 sec, detector voltage 1.3 kV and m/z range of 33-333. The components were identified by computer searching and comparing their mass spectral data with available standards and those in the WILLEY 229 mass spectra libraries.

Supercritical fluid extraction: SFE by CO2 was carried out with a laboratory-scale high-pressure extraction plant (NOVA-Swiss, Effretikon, Switzerland).

The main parts and characteristics (manufacturer specification) of the plant were as follows: a diaphragm-type compressor (up to 1000 bar), extractor with an internal volume of 200 mL (Pmax = 700 bar), separator with internal volume of 200 mL (Pmax = 250 bar) and maximum CO2 mass flow rate of approximately 5.7 kg h-1.

For each extraction test 60 g of sage with a mean diameter of 0.32 mm were charged into the extractor. The mean diameter of particles was determined by mechanical sieving. Flow rate of CO2 was 3.23x10-3 kg min-1, temperature 313 K, the total extraction time was 4 h (samples were taken every half hour). Separator conditions were pressure 15 bar and temperature 25°C.


In order to prevent thermal decomposition of sage compounds, especially thermal degradation of some volatile oil compounds, the temperature of 40°C was selected for supercritical fluid extraction with carbon dioxide (Reverchon et al., 1995; Zoran et al., 2007). The selections of the pressures ranges were based on the fact that a great change in the density and dielectric constant of CO2 occurs between pressure 80 and 300 bar (Roy et al, 2006). The pressure varied from 80 to 300 bar variations in the solvent density and the transport properties of the system were observed.

At lower solvent densities (80 bar) very low total yield of extract was observed. The increase of pressure was accompanied by a significant increase of total yield of extract. At the end of extraction process (extraction time 4 h) essential oils isolated by hydro-distillation from CO2 extracts and their contents were calculated (Table 1).

The content of sage essential oil was determined by an official procedure (Pharmacopoeia SFRJ, 1984).

The essential oil content was the highest in extract obtained at pressure 80 bar (58.79%), the contents in other essential oils varied from 29.90 to 47.87%.

CO2 extracts and essential oils were detailed identification and quantification using GC/MS and GC/FID analyses. The results of qualitative and quantitative analyses are shown in Table 2 and 3.

Table 1: Yield of CO2-extract and essential oil in extract
Image for - Supercritical Extraction of Salvia officinalis L.

Table 2: Qualitative and quantitative content of CO2-extract
Image for - Supercritical Extraction of Salvia officinalis L.

Table 3: Qualitative and quantitative content of essential oil
Image for - Supercritical Extraction of Salvia officinalis L.

The predominant component in extract obtained at 80 bar was γ-elemene (24.98%). The isoborneole (11.29%), selina-3,7 (11) diene (11.25%), 1,11-epoxyhumulene(8.99%) were detected in significant amount. The main component in extracts obtained at 100, 150, 200 and 300 bar was phyllocladene (21.99-30.64%). These extracts show high content of camphor (11.37-15.24%), selina-3,7 (11) diene (12.14-13.83%), γ-elemene (7.02-9.73%), isoborneole (6.80-9.52%).

Table 4: Essential oil content in CO2 extract and residual drug
Image for - Supercritical Extraction of Salvia officinalis L.

The main components in essential oils were α-thujone (15.63-27.38%), camphor (16.03-23.45%), isoborneole (7.94-12.11%), γ-elemene (7.46-15.52%), phyllocladene (4.19-23.37%).

In continue of investigations, the kinetics of extraction where each point of the kinetic curves obtained with the new sample of drug in extractor at pressures 80, 100 and 200 bar were studied.

After supercritical extraction for defined time, essential oil isolated by hydro-distillation from CO2 extract and residual drug (samples of sage residue after extraction). Results are shown in Table 4.

It can be seen that content of essential oil obtained from extract and residual drug (drug after extraction) decreased with pressure and time.


In this study, wild growing sage from Berkovici region were used to obtain extracts. Therewith, in order to achieve higher selectivity of the SC-CO2 extraction and thus higher antioxidant activity of the SC-CO2 extracts, no modifier were used in this study. This could also be the reason for the lower yields of antioxidant extracts reported in this study. Flavonoids, fatty acids, terpens and diterpenoids were identified in the SC-CO2 extracts of sage. The main difference between extracts and essential oils was the content of sesquiterpenes and diterpenes which are higher in the extracts.

Samples of sage were extracted by varying CO2 pressure from 80 to 300 bar. Other extraction parameters were constant: temperature 313 K, flow rate 3.23x10-3 kg min-1, mean particle diameter 0.32 mm. The composition of sage extracts and essential oils obtained by supercritical extraction is largely influenced by solvent density. Supercritical extraction by CO2 allowed optimization of the experimental conditions for selection of substances of interest, namely sesquiterpenes and diterpenes.

According to the results of this investigation, SFE offered more choices (pressures level) for the extraction of different components. On the basis of these results, it could be concluded that the extracts of the investigated sage are different.


This study was supported by the Ministry of Science and Technology the Republic of Srpska.


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