Kinetic Investigation of Rosemary Essential Oil by Two Methods: Solvent-Free Microwave Extraction and Hydrodistillation

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1 Food Anal. Methods (2012) 5: DOI /s Kinetic Investigation of Rosemary Essential Oil by Two Methods: Solvent-Free Microwave Extraction and Hydrodistillation Nacéra Tigrine-Kordjani & Brahim Youcef Meklati & Farid Chemat & Fatma Zohra Guezil Received: 30 May 2011 /Accepted: 4 August 2011 /Published online: 23 August 2011 # Springer Science+Business Media, LLC 2011 Abstract Depending on the use area of rosemary essential oil, pharmaceutical, cosmetic, or in food, the choice of both method and extraction time is fundamental, and the sample characterization is mainly based on comparing a few compounds that act as markers of a defined quality for a precise application. In the present work, the kinetic study of major components of Rosmarinus officinalis L. essential oils obtained by solvent-free microwave extraction (SMFE) and by hydrodistillation (HD) has been followed in order to determine the optimum extraction time of a defined bioactive molecule or its chemical family as well as its recommended extraction technique, either SFME or HD. The monoterpene hydrocarbons were nearly extracted completely in the first instants by SFME and progressively by HD. In contrast, the oxygenated monoterpenes were substantially extracted during the first minutes by HD and progressively by SFME. While the extraction of transcaryophyllene was achieved at the beginning with SFME, it N. Tigrine-Kordjani (*) : F. Z. Guezil Laboratoire d Analyse Organique Fonctionnelle, Faculté de Chimie, Université des Sciences et de la Technologie Houari Boumediene, El Alia, BP 32, Bab Ezzouar, 16111, Algiers, Algeria tigkor12@yahoo.fr B. Y. Meklati Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques (CRAPC), BP 248, Alger RP, 16004, Algiers, Algeria F. Chemat UMR A 408 INRA, Université d Avignon, Sécurité et Qualité des Produits d Origine Végétale, 33, rue Louis Pasteur, Avignon cedex 1, France required more than 2 h with HD. A substantial gain in the extraction time has been obtained using SFME. Even though the essential oils extracted by SFME (30 min) and HD (3 h) are qualitatively similar, the oil fractions obtained during extraction time are very different. Keywords Rosmarinus officinalis L.. Solvent-free microwave extraction. Essential oil. Hydrodistillation. Kinetic study. GC. GC-MS Introduction Rosemary (Rosmarinus officinalis L.) is an aromatic and an evergreen shrub with an intense pleasant smell. The plant, belonging to Labiatae family, grows to a height of up to 2 m and has linear leathery leaves that are sharply pointed, deep green with revolute margins, and whitish beneath. Its flowers are pale to mid-blue, mm long (Quezel and Santa 1963). It is a wild plant or cultivated in the Mediterranean region: Algeria, Spain, Morocco, Tunisia, France, and Italy, and it is widely known for its culinary and folklore medicinal uses (Flamini et al. 2002). It is the most exploited species in Algeria. It has been used in phytotherapy and currently has great potential due to the different activities of its secondary metabolites, including essential oil which has several activities: antimicrobial, spasmolytic, antioxidant, hepatoprotective, antiviral, and anticarcinogenic (Bozin et al. 2007; Fernandez-Lopez et al. 2005). Essential oil from flowers and leaves is also used externally in circulatory disorders; vapor baths are excellent in catarrh, rheumatism, and muscular affections. Extracts of R. officinalis are used in aromatherapy to treat anxiety-related conditions and to increase alertness. To improve the flavor and organoleptic properties, rosemary has been added to different types of

2 Food Anal. Methods (2012) 5: foods since ancient times. Antioxidant activity of R. officinalis hadbeenreportedtobemorepotentthansynthetic phenolic antioxidant (Herrero et al. 2010; Mc Bride et al. 2007; Mc Carthy et al. 2001). The essential oil composition of rosemary has been the subject of considerable research in recent years. It contains mainly monoterpenes and monoterpene derivatives (95 98%), the remainder (2 5%) being sesquiterpenes (Angioni et al. 2004). The principal volatile compounds in rosemary are camphor and 1,8-cineol, followed by borneol, verbenone, α-pinene, and camphene (Pino et al. 1998). Its essence showed high variations in antimicrobial and antioxidant activities. Many studies have pointed out the variability of the composition and yield of the essential oil, due to intrinsic (genetics, subspecies, and plant age) (Zaouali et al. 2010) or extrinsic factors such as climate and cultivation conditions (geographical origin) (Tigrine- Kordjani et al. 2007; Flamini et al. 2002). These variations were also correlated to the extraction process (Okoh et al. 2010; Rezzoug et al. 2005). In a previous work (Tigrine-Kordjani et al. 2006), the chemical composition of Algerian rosemary essential oils obtained using the microwave heating technique DryDist and hydrodistillation with Clevenger apparatus, the reference extraction technique according to the French standardization method AFNOR (2000), has been compared. α-pinene, camphene, limonene, camphor, and verbenone were the main components in the essential oil extracted from rosemary, but the relative amounts differed for the two extraction methods. Nowadays analytical chemistry research focuses on determining which plant constituents are responsible for each biological activity (Ramírez and Garcia-Risco 2006; Lopez et al. 2006; Thorsen and Hildebrandt 2003; Torre et al. 2001). In order to isolate from the volatile fraction substances that can find a precise application and to render the isolation strategy less laborious, one should select extracts to be investigated carefully depending on duration and extraction method. Different chemotypes of rosemary have been characterized either with a high proportion of bornyl acetate (acts against liver disease), a high cineol content (used for breathing problems), or a high level of camphor, which are known for their effects on heart tonicity (Helbert 2000). In perfumery, for example, rosemary with a high content of limonene is a component of perfumes especially for men with citrus (colognes), bush, and ferns aromatics. The objectives of the present investigation were to study the extraction kinetics of the major components of the rosemary essential oil obtained by solvent-free microwave extraction technique and to compare the results with those achieved using the hydrodistillation conventional method. Consequentially, the optimum extraction time in either SFME or HD is determined as to obtain sufficient amounts of a given essential oil s chemical family or a specific compound that presents some features in the treatment of a disease or is responsible for any of the activities. Our contribution is to determine the influence of the time parameter on the quality of the essential oil during the extraction by SFME which is a recent solvent-less method (green chemistry) and HD as conventional method. Experimental Plant Material Rosemary (R. officinalis L.) was collected from the north of Algeria. In order to avoid the influence of vegetative cycle on the composition of the essential oil, it was gathered for all extractions over a short period of time (during November 2009) under the same conditions from the Institute National Agronomic experimental plantation (Algiers, Algeria). Only fresh aerial plant material was employed. It was authenticated by the Botanic Department of the National Agronomic Institute, Algiers, Algeria. Microwave Process SFME The microwave extraction process was carried out in a microwave laboratory oven as described in Lucchesi et al. 2004; Chematetal.2004), at atmospheric pressure; 200 g of aerial parts of fresh rosemary was heated using a fixed power of 1,000 W without adding any solvent or water. The extraction time is fixed at 30 min when no more essential oil was obtained; however, the kinetic survey was followed at different extraction times of 3, 5, 10, 15, 20, and 30 min. The essential oils were collected, dried over anhydrous sodium sulfate, and stored at 4 C until subsequent analysis. The extractions were performed at least three times, and the mean values were reported. Hydrodistillation Process HD For each extraction, 200 g of fresh rosemary composed of stems, leaves, and flowers was submitted to hydrodistillation with a Clevenger-type apparatus (Clevenger 1928), according to the European Pharmacopoeia and extracted with 2 L of water. While in the discontinuous process, separated extractions were performed at different times: 30 min, 1 h, 2 h, and 3 h; in the continuous process and during the same extraction, successive withdrawals of essential oil fractions were made at different times: 5, 10, 45, 75, 120, 150, and 180 min. The essential oil fractions were collected, dried over anhydrous sodium sulfate, and stored at 4 C until subsequent analysis. The extractions were performed at least three times, and the mean values were reported.

3 598 Food Anal. Methods (2012) 5: Table 1 Chemical composition of R. officinalis essential oils obtained by SFME (30 min) and HD (3 h) No. Compounds a RI b RI c SFME% HD% Monoterpene hydrocarbons Tricyclene 921 1, α-pinene 937 1, ,53 3 Camphene 949 1, Verbenene 953 1, β-pinene 972 1, β-myrcene 989 1, I-Phellandrene 1,000 1, δ-3-carene 1,005 1, α-terpinene 1,012 1, Para-cymene 1,023 1, Limonene 1,027 1, γ-terpinene 1,052 1, α-terpinolene 1,080 1, Oxygenated monoterpenes Linalool 1,105 1, Chrysantenone 1,116 1, α-campholenal 1,120 1, Camphor 1,146 1, Pinocarvone 1,160 1, Borneol 1,166 1, Terpinen-4-ol 1,177 1, γ-campholenol 1,181 1, α-terpineol 1,189 1, Myrtenol 1,201 1, tr 24 Verbenone 1,205 1, Trans-carveol 1,226 1, Citronellol 1,234 1, Carvone 1,244 1, Carvotanacetone 1,247 1, Piperitone 1,252 1, Geraniol 1,258 1, Carvacrol 1,303 2, Eugenol 1,353 2, Methyl eugenol 1,397 2, Geranyl acetone 1,444 1, Sesquiterpene hydrocarbons Trans-caryophyllene 1,408 1, α-humulene 1,442 1, E-β-Farnesene 1,447 1, α-acoradiene 1,454 1, α-curcumene 1,473 1, α-farnesene 1,500 1, Oxygenated sesquiterpenes Caryophyllene oxide 1,564 1, Humulene epoxide <1,2-> 1,587 2, β-caryophylla-3(8)[13] diene 5-ol 1,641 2, β-sesquicyclogeraniol 1,648 2, α-bisabolol 1,673 2,

4 Food Anal. Methods (2012) 5: Table 1 (continued) No. Compounds a RI b RI c SFME% HD% Other oxygenated compounds , ,6-Octadienoic acid-3,7-dimethyl methyl ester 1,274 1,610 tr Bornyl acetate 1,281 1, Methyl-jasmonate 1,635 2, Extraction time (min) Yield (%) 0.56± ±0.09 % Total oxygenated compounds % Total non-oxygenated compounds % Major components d tr traces a Essential oil compounds sorted by chemical families and percentages calculated by GC-FID on non-polar HP5MS capillary column b Retention indices with respect to C 5 C 28 n-alkanes calculated on non-polar HP5MS capillary column c Retention indices with respect to C 5 C 28 n-alkanes calculated on polar Carbowax -PEG capillary column d α-pinene, camphene, limonene, p-cymene, camphor, verbenone, α-terpineol, linalool, borneol, and trans-caryophyllene GC and GC-MS Analyses A gas chromatography flame ionization detector (GC-FID) system (Agilent, CA, USA, 2000) was used for gas chromatography analysis fitted with a fused-silica-capillary column containing a non-polar stationary phase HP5MS (30 m 0.25 mm 0.25 μm film thickness). The column temperature program was 60 C for 8 min then was increased at 2 C/min to 250 C and held at 250 C for 15 min. A volume of 0.2 μlwas injected into the splitless GC inlet held at 250 C for all samples. The carrier gas was nitrogen flowing through the column at 0.3 ml/min. The temperature of the flame ionization detector was 320 C. The essential oils were also analyzed by a gas chromatograph coupled to a mass spectrometer (GC-MS, Agilent, Palo Alto, CA, USA, 2000) using two fused-silica-capillary columns with different stationary phases. The non-polar column was HP5MS (30 m 0.25 mm 0.25 μm film thickness), and the polar one was a Stabilwax consisting of Carbowax PEG (60 m 0.2 mm 0.25 μm film thickness). GC-MS was operated using the following conditions: carrier gas, He; flow rate, 0.3 ml/min; splitless mode; injected volume for all samples, 0.2 μl; injection temperature, 250 C; the oven temperature program was 60 C for 8 min increased at a rate of 2 C/min to 250 C and held at 250 C for 15 min; the ionization mode used was electronic impact at 70 ev. The homologous n-alkanes series C 5 C 28 injected in GC and GC-MS under the same conditions as the essential oils were used to calculate the retention indices. Relative amount of individual components is based on peak areas obtained without FID response factor correction. Three replicates were performed for each sample. The average of these three values and the standard deviation were determined for each identified component. The component identification was established by comparison of the mass spectral fragmentation patterns with those stored in the database NIST 2006 and Wiley 7. The retention indices of the essential oil constituents compared with those of the published index data (Adams et al. 2007) confirmed the identification. Results and Discussion Extraction Extraction time of 30 min with SFME provides yields (0.56±0.05%) comparable to HD yields (0.57±0.09%) obtained after 3 h. It is worth noting that with SFME, the Fig. 1 Yield profiles as a function of time for extraction processes SFME (a) and HD(b)of essential oil from aerial parts of R. officinalis L.

5 600 Food Anal. Methods (2012) 5: Table 2 Kinetics major compounds SFME Time of extraction (min) α-pinene Camphene Para-cymene Limonene % Total of monoterpene hydrocarbons Linalool Camphor Borneol α-terpineol Verbenone % Total of oxygenated monoterpenes Trans-caryophyllene time to reach the extraction temperature (100 C) for getting the first essential oil droplet is only 2.5 min compared to 45 min for HD, and all extraction times cited above were held at the beginning of the actual extraction. The analyses by GC (FID) and GC-MS have shown that the essential oils extracted by both methods (SFME 30 min and HD 3 h ) had similar qualitative chemical compositions but were different quantitatively. The identification of 48 compounds enabled the detection of most volatile active compounds in rosemary essential oil such as α-pinene, camphor, verbenone, borneol, linalool, para-cymene, and camphene, but their proportions fairly depend on the isolation technique. Substantial amounts of oxygenated compounds (37.89% versus 30.48%) and lower amounts of monoterpene hydrocarbons (60% versus 67.33%) are present in the essential oil of rosemary extracted by SFME in comparison with HD (Table 1). In both cases, more than 80% of the essential oil chemical compositions are represented by ten compounds. These compounds are α-pinene, camphene, limonene, and para-cymene as monoterpene hydrocarbons; camphor, verbenone, α-terpineol, linalool, and borneol as oxygenated monoterpenes; and trans-caryophyllene as sesquiterpene. Therefore, the main chemical families of R. officinalis L. essential oils are monoterpene hydrocarbons, oxygenated monoterpenes, and sesquiterpene. Kinetics Figure 1a depicts the yields according to the extraction time in the microwave process. Three phases are observed, the first one (step 1) which lasts 5 min, corresponds to an increasing curve; this short time is sufficient to extract 75% of the total yield characterizing expansion and release of Table 3 Kinetics major compounds HD in continuous process Essential oil fraction between 0 5 min 5 10 min min min Min min min min α-pinene Camphene Para-cymene Limonene % Total of monoterpene hydr Linalool Camphor Borneol α-terpineol Verbenone % Total of oxyg monoterpenes Trans-caryophyllene

6 Food Anal. Methods (2012) 5: Table 4 Kinetics major compounds HD in discontinuous process Extraction time 30 min 1 h 2 h 3 h α-pinene Camphene Para-cymene Limonene % Total monoterpene hydrocarbons Linalool Camphor Borneol α-terpineol Verbenone % Total Oxy. monoterpenes Trans-caryophyllene essential oil from exogenous secreting glands. The second phase located between 5 and 20 min (step 2) where nearly 25% of the global yield is extracted concerns the diffusion of the essential oil of the endogenous sites which are more difficult to be damaged than the most external ones. The last stage highlighted by a horizontal line (step 3) indicates that the essential oil is completely extracted from the plant. Figure 1b shows the variation of the yield according to the extraction time in the hydrodistillation method; the curve can be divided into three stages. During the first phase (step 1) in which the duration is 30 min, 72% of the total yield is extracted progressively. This stage characterizes the first quantities extracted, located at the surface of vegetable particles. The second phase is located between 30 and 150 min (step 2), where the quantity that remained, almost equal to 28% of the total yield, is extracted slowly during 2 h. In the last stage represented by a horizontal line (step 3), the essential oil is completely extracted from the plant. To assess the influence of extraction time on the essential oil composition, all fractions collected during the study of the yield kinetics of both extraction techniques SFME and HD were analyzed by gas chromatography with a flame ionization detector GC-FID and by gas chromatography coupled with mass spectrometry GC-MS. We were able to monitor the content of the ten major components by referring to the files of the chromatograms obtained by GC-FID. Kinetics Microwave Process by GC and GC-MS Table 2 shows that the content of major components monoterpene hydrocarbons of volatile oil of R. officinalis extracted by SFME increases from 3 (65.15%) to 5 min (66.54%) and decreases gradually afterwards, till it reaches 43.31% at 30 min. The highest content for α-pinene is 50.38%, and for limonene, it is 6.82% at 5 and 3 min of the extraction, respectively. This indicates that the monoterpene hydrocarbons require a very short time to be extracted by microwave process. The extraction of oxygenated monoterpenes is progressive over time. At 3 min, the content is 16.33%. The latter continues to increase until it reaches a percentage of 36.76% in 30 min of extraction with camphor (12.86%) as major component. The trans-caryophyllene, relatively heavy sesquiterpene compound, is extracted in the first 3 min. Its extraction is phased over time until it reaches a percentage of 3.89% in 30 min. Kinetics Hydrodistillation in Continuous Process by GC and GC-MS Between 0 and 10 min, the content of monoterpene hydrocarbons decreases (Table 3). It characterizes the first quantities extracted and corresponds to exogenous sites that are easily damaged by temperature within minutes. Then, an increase in their extraction between 10 and 75 min is observed, which corresponds to the gradual breakdown of the glands and oleiferous receptacles endogenous sites. In the oil fraction from 45 to 75 min, the content of monoterpene hydrocarbons is the highest; it reaches a maximum at 79.34% with α-pinene at 57.74% and limonene at 8.67%. After 2 h of extraction, the content of monoterpene hydrocarbons is only 11.56% and 10.50% in the oil fractions at and min extraction times, respectively. The highest content of para-cymene is 4.47% in the oil fraction from 30 to 45 min. From 0 to 120 min, the oxygenated monoterpene content decreases gradually. The content is maximal at 5 min of extraction and reaches a height peak of 24.29% where the content of camphor is 10.10%. The total content is minimal during the period from 75 to 120 min with a height of 1.43%. Between 120 and 180 min, a slight increase from 1.43% to 2.06% is noticed; this is due to the depletion of

7 602 Food Anal. Methods (2012) 5: monoterpene hydrocarbons, considered as major compounds of the essential oil. The trans-caryophyllene extraction is so low between 0 and 120 min; its height is between 0.4% and 1.19%. From 120 till 180 min, the increase is very important; it reaches a height of 27.2%, and a considerable extraction of other sesquiterpenes: α-humulene, α-curcumene, and oxygenated sesquiterpenes such as caryophyllene oxide are observed at this stage. Kinetics Hydrodistillation in Discontinuous Process by GC and GC-MS Table 4 shows that the content of major components monoterpene hydrocarbons extracted by hydrodistillation in discontinuous process increases from 30 min to 2 h and then decreases at 3 h. The highest content, i.e., 60.08%, was recorded at 2 h. The content of oxygenated monoterpenes decreases over time. It reaches a maximum height of 29.41% in 30 min and a minimum height of 21.78% in 3 h. The extraction of trans-caryophyllene is almost constant in the range from 30 min to 2 h. It increases slightly after 2 h. Its maximum height is 0.72%. Both hydrodistillations in continuous and discontinuous processes lead to similar results. The monoterpene hydrocarbons are extracted progressively during the first 2 h of extraction. The content of oxygenated monoterpenes is the most important at the first minutes of the extraction duration and decreases gradually until depletion of the plant s essential oil. The trans-caryophyllene s extraction, compound relatively heavy of sesquiterpene family, is low during the first 2 h and becomes important after 2 h of extraction. Conclusion The monoterpene hydrocarbons represented by α-pinene, camphene, limonene, and para-cymene are nearly extracted completely in the first instants by the SFME method, whereas the oxygenated compounds represented by camphor, verbenone, linalool, borneol, and α-terpineol are extracted progressively throughout the extraction process of this method. Inversely, the oxygenated monoterpenes are extracted substantially during the first minutes by hydrodistillation, and the monoterpene hydrocarbons are extracted progressively during the first 2 h of the HD extraction. Trans-caryophyllene which represents the sesquiterpene family is extracted by HD at an important quantity only after 2 h, whereas by SFME, it is already extracted at the beginning of the extraction. The SFME technique is recommended for samples of essential oil rich in monoterpene hydrocarbons considered as bioactive molecules. We showed in this study that with SFME, 5 min was found to be sufficient to allow a sample extraction containing 66.54% of α-pinene, camphene, limonene, and para-cymene, while extracting a sample containing 63.08% of these compounds is obtained after an extraction time of 2 h by hydrodistillation technique. However, in the case of essential oil samples rich in oxygenated monoterpenes as active compounds, the conventional method requires only 5 min to offer a sample containing 24.29% of linalool, camphor, borneol, α- terpineol, and verbenone while the SFME process provides a sample containing 36.76% of these compounds in 30 min. Therefore, the choice of either of these extraction methods depends on the desired quality of the essential oils. References Adams RP (2007) Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing Corporation, Carol Stream Angioni A, Barra A, Cereti E, Barile D, Coisson JD, Arlorio M, Dessi S, Coroneo V, Cabras P (2004) Chemical composition, plant genetic differences, antimicrobial and antifungal activity investigation of the essential of Rosmarinus officinalis L. J Agric Food Chem 52: Bozin B, Mimica-Dukic N, Samojlik I, Jovin E (2007) Antimicrobial and antioxidant properties of rosemary and sage Rosmarinus officinalis L. and Salvia officinalis L., Lamiaceae essential oils. J Agric Food Chem 55: Chemat F, Smadja J, Lucchesi ME (2004) Brevet Européen, EP A1 Clevenger J (1928) Apparatus for volatile oil determination, description of new type. American Perfumer & Essential Oil Review: Fernandez-Lopez J, Zhi N, Aleson-Carbonell L, Perez-Alvarez JA, Kuri V (2005) Antioxidant and antibacterial activities of natural extracts: application in beef meatballs. Meat Sci 69: Flamini G, Cioni PL, Morelli I, Macchia M, Ceccarini L (2002) Main agronomic-productive characteristics of two ecotypes of Rosmarinus officinalis L. and chemical composition of their essential oils. J Agric Food Chem 50: Helbert S (2000) Revue de formation médicale continue en homéopathie, LyLach No. 18 Herrero M, Plaza M, Cifuents A (2010) Green processes for the extraction of bioactives from rosemary: chemical and functional characterization via ultra-performance liquid chromatography-tandem mass spectrometry and in-vitro assays. J Chromatogr A 1217: Lopez P, Huerga MA, Batlle R, Nerin C (2006) Use of solid phase microextraction in diffusive sampling of the atmosphere generated by different essential oils. Anal Chim Acta 559: Lucchesi ME, Chemat F, Smadja J (2004) Solvent free microwave extraction of essential oil from aromatic herbs: comparison with conventional hydro-distillation, short Communication. J Chromatogr A 1043: Mc Bride NTM, Hogan SA, Kerry JP (2007) Comparative addition of rosemary extract and additives on sensory and antioxidant properties of retail packaged beef. Int J Food Sci Technol 42: Mc Carthy TL, Kerry JP, Kerry JF, Lynch PB, Buckley DJ (2001) Evaluation of the antioxidant potential of natural food/plant

8 Food Anal. Methods (2012) 5: extracts as compared with synthetic antioxidants and vitamin E in raw and cooked pork patties. Meat Sci 57:45 52 Okoh OO, Sadimenko AP, Afolayan AJ (2010) Comparative evaluation of the antibacterial activities of the essential oil of Rosmarinus officinalis L. obtained by hydrodistillation and solvent free microwave extraction methods. Food Chem 120: Pino JA, Estarrón M, Fuentes V (1998) Essential oil of rosemary (Rosmarinus officinalis L.). J Essent Oil Res 10: Quezel P, Santa S (1963) Nouvelle flore de l Algérie et des régions désertiques méridionales. Editions du centre national de la recherche scientifique Ramírez P, Garcia-Risco MR (2006) Isolation of functional ingredients from rosemary by preparative-supercritical fluid chromatography (Prep-SFC). J Pharm Biomed Anal 41: Rezzoug SA, Boutekedjiret C, Allaf K (2005) Optimization of operating conditions of rosemary essential oil extraction a fast controlled pressure drop process using response surface methodology. J Food Eng 71:9 17 Thorsen MA, Hildebrandt KS (2003) Quantitative determination of phenolic diterpenes in rosemary extracts: aspects of accurate quantification. J Chromatogr A 995: Tigrine-Kordjani N, Meklati BY, Chemat F (2006) Microwave Dry distillation as an useful tool for extraction of edible essential oils. Int J Aromather 16: Tigrine-Kordjani N, Chemat F, Meklati BY, Montury M (2007) Relative characterization of rosemary samples according to their geographical origins using microwave-accelerated distillation, solid-phase microextraction and Kohonen self-organizing maps. Anal Bioanal Chem 389: Torre J, Lorenzo MP, Martinez-Alcazar MP, Barbas C (2001) Simple high-performance liquid chromatography method for α- tocopherol measurement in Rosmarinus officinalis leaves: new data on α-tocopherol content. J Chromatogr A 919: Zaouali Y, Bouzine T, Boussaid M (2010) Essential oils composition in two Rosmarinus officinalis L. varieties and incidence for antimicrobial and antioxidant activities. Food Chem Toxicol 48: All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.