Simultaneous Distillation–Extraction of Essential Oils from Rosmarinus ofﬁcinalis L.

: The present study describes a procedure to isolate essential oils from Rosmarinus ofﬁcinalis L. using simultaneous distillation–extraction (SDE). Rosmarinus ofﬁcinalis L. can be used for medicinal purposes, as well in the cooking and cosmetics industries. SDE technique extraction combines a steam distillation combined with a continuous extraction using a solvent or a co-solvent mixture, providing faster extractions with low extraction solvent volumes. The effect of the solvent nature and the extraction time on the simultaneous distillation–extraction efﬁciency was evaluated. The best performance was achieved using pentane as a solvent for 1 h of extraction. The essential oils obtained by simultaneous distillation–extraction extracts were analyzed by gas chromatography with ﬂame ionization detection (GC-FID). Extraction efﬁciencies ranged from 40 to 70% for the majority of the compounds tested, and the precision (measured by the relative standard deviation) varied between 6 and 35%. Among the compounds analyzed the most abundant in the Rosmarinus ofﬁcinalis L. sample were 1,8-cineole, (-) –borneol, α -pinene, (S)-(-)- α –terpineol, (-)-bornyl acetate, linalool, and 2,2,6-trimethylcyclohexanone. The SDE method proved to be a suitable option for obtaining extracts free from cuticular waxes or chlorophylls.


Introduction
Rosmarinus officinalis L. is an aromatic shrub with an intense, pleasant smell belonging to the Lamiaceae family [1][2][3]. It is indigenous to the Mediterranean region, cultivated mainly in Spain, Morocco, and Tunisia [1], but also China, India, Algerian, North America, Northern Europe, and England [4]. In Portugal, it is an abundant species, mainly in the North East, Centre, and South of the country mainland.
However, in the innermost regions, it is possible to find more abundance. The flowering season is very long and gradual, from April to August, but often, it flowers all year long. Due to its rusticity, it grows in every soil type, but it prefers a sandy, arid, calcareous, and humus-poor soil [1].
Rosmarinus officinalis L. has been used since ancient times for medicinal purposes and is known for its antiseptic, anti-rheumatic, anti-inflammatory, and antispasmodic properties. R. officinalis extracts also exhibit hepatoprotective, anti-diabetic, anti-ulcerogenic, and antidepressant effects [5,6]. This plant can be used fresh, dried, or as a tea infusion, for cooking purposes as flavoring agents, in the preservation of foods, and cosmetics [6,7].
Essential oils extracted from Rosmanirus Officinalis L. are widely used in the cosmetics and perfumery industry and also aromatherapy, in particular by therapeutic techniques such as massage, inhalation, or bath. They are also used as ingredients in the pharmaceuti-2 of 10 cal industry to flavor oral forms, to perfume dermo-pharmaceutical preparations, and to ensure the preservation of pharmaceutical forms [8].
Due to their broad antimicrobial activities, the essential oils of Rosmanirus Officinalis L. have preservative potential for the food and cosmetic industries [9].
Essential oils have a complex composition, containing a few dozens to several hundred constituents, especially hydrocarbons (terpenes and sesquiterpenes) and oxygenated compounds (alcohols, aldehydes, ketones, acids, phenols, oxides, lactones, acetals, ethers, and esters). Both hydrocarbons and oxygenated compounds are responsible for the characteristic odors and flavors [10]. Many studies have pointed out the variability of the composition and the yield of the essential oil due to intrinsic (genetics, subspecies, and plant age) or extrinsic factors, such as climate and cultivation conditions (geographical origin) or isolation methods [1].
This variation of the composition is usually more quantitative than qualitative, and due to that, essays performed with essential oils should always provide a biological characterization of the plant material and the oil's phytochemical profile, enabling the reproducibility and accuracy of data.
Essential oils can also be microencapsulated for use in cosmetics and personal healthcare products. In cosmetic products, essential oils (EOs) play a major role as fragrance ingredients. They can optimize its proprieties and preservation, as well as the marketing image of the final product. Microencapsulation of EOs can protect and prevent the loss of volatile aromatic ingredients and improve the controlled release and stability of these core materials [11][12][13].
Extraction techniques based on compounds solubility involve a direct contact of the sample with the solvent (e.g., supercritical fluid extraction) or with the adsorbent (e.g., solid-phase microextraction in the direct extraction mode), leading to a co-extraction of heavy components which pollutes gas chromatograph injectors.
Volatility-based techniques do not exhibit such a drawback; however, they either yield high water volumes containing a low concentration of the volatiles (hydrodistillation, steam distillation), or they isolate the most volatile fraction. Only a few methods take advantage of both properties [4,20].
Simultaneous distillation-extraction (SDE), introduced in 1964 by Likens and Nickerson, has been successfully applied in the extraction of essential oils, aromatic compounds, and other volatile products from several matrices. This technique has usually been considered superior to classical ones, such as distillation or solvent extraction, once it combines steam distillation with continuous extraction with a solvent or a mixture of solvents [21].
This one-step isolation-concentration technique allows a dramatic time saving over the separated operation and, because of their continuous recycling, a great reduction in treated volumes of liquids [20].
This technique does not require a clean-up step. Moreover, the extracts obtained by SDE are free from non-volatile materials such as cuticular waxes or chlorophylls. The SDE enables high extraction efficiencies associated with high reproducibility. This technique has also been used to analyze volatile compounds in several matrices [21].

Sample
The sample analyzed was Rosmarinus officinalis L., collected on 10 March 2018, in the Portuguese region of Vila Real.

Apparatus and Material
Gas chromatography was performed using a Bruker 430-GC gas chromatograph (GC) equipped with a Flame Ionization Detector (FID). A BR-1MS column from Bruker was used (15 m length, 0.25 mm internal diameter, 0.25 µm film thickness). The GC oven was programmed (50 • C for 1 min, raised a rate of 50 • C/min until 280 • C and then maintained for 1 min). Injections were performed in split mode (1:10 ratio) using a 0.5 µL syringe (SGE), and the injection volume was 0.2 µL. Helium was used as carrier gas at a constant flow of 1 mL/min.
Extractions were performed in a Simultaneous Distillation Extraction (SDE) using a Likens-Nickerson apparatus.

Extraction
For the extraction of samples, 25 g of fresh plant grinded into a fine paste using a mortar and pestle were placed in the aqueous flask together with 250 mL of distilled water, using a method adapted from [21].
The preparation of the organic phase included 50 mL of solvent (either n-Pentane or Chloroform) was introduced. The time of extraction was optimized from 30 to 120 min. The solvent was removed in a rotating evaporator (Buchi Rotavapor E-210 rotary evaporator) at room temperature. The extract was washed with 4 portions of 1 mL of ethanol and transferred to a vial. Ethanol was then evaporated under a gentle stream of nitrogen (approximately 1 L/min) and the extract was reconstituted with 1 mL of ethanol.

Method Optimization and Validation
Individual compounds were identified by the retention time using the injection of individual standards.
The calibration curves for all the essential oils were obtained by the internal standard method, using acetophenone as the internal standard, at a constant concentration of 39 mg/L. For each oil, seven concentration level standards were prepared in ethanol. The prepared samples were injected in triplicate, as well as the respective calibration standards. The injections in the GC were performed manually.

Results and Discussion
After identifying chromatographic peaks (Figure 1), calibration was performed. The results of the retention times, the concentration range, and the correlation coefficient are presented in Table 2.
The solvent was removed in a rotating evaporator (Buchi Rotavapor E-210 rotary evapo rator) at room temperature. The extract was washed with 4 portions of 1 mL of ethano and transferred to a vial. Ethanol was then evaporated under a gentle stream of nitroge (approximately 1 L/min) and the extract was reconstituted with 1 mL of ethanol.

Method Optimization and Validation
Individual compounds were identified by the retention time using the injection o individual standards.
The calibration curves for all the essential oils were obtained by the internal standar method, using acetophenone as the internal standard, at a constant concentration of 3 mg/L. For each oil, seven concentration level standards were prepared in ethanol. Th prepared samples were injected in triplicate, as well as the respective calibration stand ards. The injections in the GC were performed manually.

Results and Discussion
After identifying chromatographic peaks (Figure 1), calibration was performed. Th results of the retention times, the concentration range, and the correlation coefficient ar presented in Table 2.   Two extraction solvents were tested: n-Pentane and Chloroform. For these two solvents, three different times of extraction were tested: 30, 60, and 120 min. Extraction efficiency was evaluated by extracting 100 µL of a highly concentrated oil mixture. Extraction efficiency was calculated as the ratio between the concentration determined by the analysis of the spiked extract and the amount of compound spiked to the water. The concentration of each compound was calculated by the ratio between the amount of compound quantified in the extract divided by the extraction efficiency for that compound and divided by the weight of the sample extracted.
Extraction efficiency results are presented in Figures 2 and 3. Two extraction solvents were tested: n-Pentane and Chloroform. For these two solvents, three different times of extraction were tested: 30, 60, and 120 min. Extraction efficiency was evaluated by extracting 100 μL of a highly concentrated oil mixture. Extraction efficiency was calculated as the ratio between the concentration determined by the analysis of the spiked extract and the amount of compound spiked to the water. The concentration of each compound was calculated by the ratio between the amount of compound quantified in the extract divided by the extraction efficiency for that compound and divided by the weight of the sample extracted.
Extraction efficiency results are presented in Figures 2 and 3.   Less extraction was observed for α-pinene as the time of extraction increased. This should be caused by the losses by evaporation due to the volatility of this compound, once it is the one with the lowest boiling point and due to the fact that the boiling point of chloroform is superior to n-pentane and as such, evaporation occurs at higher temperatures. Furthermore, α-pinene also has a lower solubility in chloroform than in n-pentane.
Since extraction efficiencies were similar for both solvents, n-pentane was chosen due to the environmental impact of chloroform. The time of extraction chosen was 30 min once α-pinene was detected with this extraction time, and extraction efficiency did not increase with time for the other compounds.
Extraction efficiency was tested using n-Pentane as the extraction solvent and with 30 min of extraction time. Extraction efficiency results are presented in Table 3 and show very good accuracy, being the relative standard deviation (RSD (%)) lower than 35% for all compounds. Excluding α-pinene, extraction efficiencies were high, on average from 53 to 88%.  Trimethylcyclohexanone  52  52  60  48  53  9  1,8-Cineole  52  53  62  50  54  10  Linalool  93  95  88  65  85  Less extraction was observed for α-pinene as the time of extraction increased. This should be caused by the losses by evaporation due to the volatility of this compound, once it is the one with the lowest boiling point and due to the fact that the boiling point of chloroform is superior to n-pentane and as such, evaporation occurs at higher temperatures. Furthermore, α-pinene also has a lower solubility in chloroform than in n-pentane.
Since extraction efficiencies were similar for both solvents, n-pentane was chosen due to the environmental impact of chloroform. The time of extraction chosen was 30 min once α-pinene was detected with this extraction time, and extraction efficiency did not increase with time for the other compounds.
This technique congregates the advantages of solvent-based and hydrodistillation extractions. A particularly high concentration factor and low solvent volume were used. Acceptable reproducibility and high extraction efficiencies were obtained.

Conclusions
A method of analysis of essential oils from Rosmarinus officinalis L., using simultaneous distillation-extraction followed by analysis by gas chromatography, was validated.
Extraction efficiencies for most compounds tested ranged from 40 to 70%. Extraction efficiencies were fairly reproducible (RSD between 6 and 35%).
Analysis by SDE-GC/FID enhances high extraction efficiencies and reasonable reproducibility with low solvent volume used. Extracts free from cuticular waxes or chlorophylls allow a good chromatographic separation. The results are very similar to those obtained by other authors, using other techniques [5,6,[12][13][14][15].
This technique congregates the advantages of solvent-based and hydrodistillation extractions. A particularly high concentration factor and low solvent volume were used. Acceptable reproducibility and high extraction efficiencies were obtained.

Conclusions
A method of analysis of essential oils from Rosmarinus officinalis L., using simultaneous distillation-extraction followed by analysis by gas chromatography, was validated.
Extraction efficiencies for most compounds tested ranged from 40 to 70%. Extraction efficiencies were fairly reproducible (RSD between 6 and 35%).
Analysis by SDE-GC/FID enhances high extraction efficiencies and reasonable reproducibility with low solvent volume used. Extracts free from cuticular waxes or chlorophylls allow a good chromatographic separation.