ОРИГИНАЛЬНЫЕ СТАТЬИ =
УДК 543
STUDY ON THE STABILITY OF SUPERCRITICAL FLUID EXTRACTED ROSEMARY (ROSMARINUS OFFCINALIS L.) ESSENTIAL OIL © 2010 Sibel Irmak, Kemal Solakyildirim, Arif Hesenov, Oktay Erbatur
Çukurova University, Department of Chemistry, Arts and Sciences Faculty Balcali, Adana 01330 Turkey Received 30.03.2009; in final form 02.10.2009
The aim of this study was to examine the influence of storage conditions and duration on composition and antioxidant activity of supercritical fluid (SCF) extracted essential oil of rosemary (Rosmarinus officinalis L.). Supercritical extraction was carried out sequentially by using SCF carbondioxide in the first two steps and with 5% ethanol as entrainer in the third step. The compositions of the extracts were determined by gas chro-matography/mass spectrometry. The total phenolics were analyzed using Folin-Ciocalteau assay. Antioxidant activities of the extracts were tested by в-carotene-linoleic acid bleaching method.
The extracts stored at 4°C in the dark for 14 weeks were associated with slight changes in their composition. However, storage under indirect day light at room temperature caused considerable changes in the compositions of the oils due to the chemical transformations in some of their components. Both the total phenolic contents and the antioxidant activities were significantly decreased after storage.
Key words: supercritical fluid extrzction, Rosmarinus offcinalis L.
Food safety is becoming an increasingly significant health issue in the food industry since noticeably increased cases offoodborne illnesses have been reported in many countries over the past few decades [1]. Besides, the toxicological aspects of using synthetic antioxidants and antimicrobials in foods on long-run basis are being questioned and this results in the formation of negative consumer perception which ultimately brings restriction of their usage as food additives. Therefore, extensive research is being conducted to adopt some natural antioxi-dants and antimicrobials into foods, preferably by incorporating into food packaging material, or by binding to the surface either permanently or temporarily, where in the latter case, the antioxidant and/or antimicrobial agents are slowly released into the packed food during the shelflife period [2—4]. In this aspect, plant-derived natural antioxidant and antimicrobial compounds as replacements for synthetic food additives have garnered a great deal of attention because of their food-preserving and health-promoting properties [5, 6].
Natural plant extracts from oregano, sage, rosemary, thyme, clove, pimento, etc. were reported to possess antimicrobial and antioxidative properties, which were either comparable to or greater than those of butylated hy-droxyanisole (BHA) and butylated hydroxytoluene (BHT) [7—10]. The antioxidant and antimicrobial properties of these extracts have been mostly attributed to the presence of phenolic compounds such as flavonoids and phenolic acids in their essential oil fractions [11]. The phenolic compounds are reported to have potential health benefits such as reducing the risk of cardiovascular disease and cancer [12].
Rosemary (Rosmarinus officinalis L.) is an evergreen plant native to Mediterranean region, that is known to exhibit antioxidant and antimicrobial activities. Comparatively, rosemary extracts have been reported to have the highest antioxidant activity which is associated with the presence of several phenolic diterpenes (carnosic acid, carnosol, rosmanol, rosmariquinone and rosmarid-iphenol), flavones (apigenin, hesperetin, genkwanin or cirsimaritin) and phenolic acids (caffeic acid and ros-marinic acid) [13]. The amount of these individual components present in the extracts are dependent on geographic location of the plant grown and the harvesting time [14, 15].
Supercritical fluid extraction is an effective technique for extraction of thermo labile, oxygen and light sensitive compounds from plants since this process takes place at reduced temperature in the absence of light. Separation of the extract from the solvent is easy in this technique, and possible residues do not cause any risk for human health. Also, it is possible to separate the extracts into several fractions by changing the extraction conditions such as pressure and temperature [14, 16]. A sequential extraction of plant materials may lead to release the potential antioxidant and antimicrobial compounds efficiently and thus helps to get antioxidant rich extracts from different plant sources.
In this study, the product compositions and antioxi-dant activities of rosemary essential oils which were sequentially extracted from plant leaves using supercritical fluid extraction technique were investigated both in the fresh forms and following storage under different conditions. Although the antioxidant activity profiles of essen-
tial oils of spices and herbs have been reported extensively, literature on their stability during storage conditions is scarce. Therefore, the present study was designed to determine the influence of storage conditions and duration on composition and antioxidant activity of supercritical fluid extracted rosemary essential oils. The findings of this study would be useful in widespread commercial applications of natural bioactive compounds in food and pharmaceutical industry.
EXPERIMENTAL
Samples. Rosemary samples used in this study were collected in November 2006 in the campus area of Quku-rova University which is located in southern part of Turkey. Rosemary leaves were dried at room temperature and ground in a coffee grinder. The ground samples which contained 7.38 ± 0.07% moisture were packed in plastic bags, protected from light and stored at 4°C for a maximum of one week till being used in extraction.
Extraction of rosemary. The extraction of rosemary samples was carried out in a 12L-pilot scale 316 stainless steel high pressure reactor (Parr Instrument Co., Moline, IL) with a temperature controller system. 2000 g of sample (7.38% moisture) was placed in the reactor and temperature was set at 40°C. The reactor was pressurized with CO2 using ISCO 260D pump (Isco Inc., Lincoln, Nebraska). When the system reached the desired pressure, it was maintained at this condition for one hour and then extract collection was started. A three-step sequential extraction was performed to extract and fractionate bioactive compounds from the material. First, the pressure was 1450 psi at 40°C and after keeping one hour at this condition, extract was collected for approximately three hours. Then, the pressure was increased to 3000 psi at 40°C and extract collection was started after one hour and continued for almost three hours duration. In the third step, 5% ethanol was added into the remained solid material and the reactor was pressurized to 2800 psi at 55°C. It was kept for one hour at this condition and extract collection was performed for three hours. The extraction yields in all steps were determined gravimetrical-ly by measuring the weights of the collection flasks before and after extract collection. The essential oils collected from all above steps were divided equally into two parts and stored in glass flasks closed with tight stoppers. One set of samples were kept under indirect day light at room temperature (24 ± 1°C) and the flasks of other set of samples were covered by aluminium foil and stored at 4°C for 14 weeks. Both sample sets were analyzed and tested by using the same methods. Antioxidant activities of the extracts were assayed following 0, 2, 4, 8 and 14 weeks of storage. GC-MS and HPLC analysis were performed following one and fourteen weeks after storage.
Analytical procedures. GC-MS analysis. Extract compositions were determined by a Thermo Finnigan Trace Gas Chromatograph and Mass Spectrometer (GC-MS) using Thermo TR-5MS capillary column (60 m x x 0.25 mm ID x 0.25 ^m film thickness). The samples
were injected to GC-MS in silylated and nonsilylated forms. The silylation was performed with N,O-bis-(trim-ethylsilyl)trifluoroacetamide (BSTFA, Aldrich) reagent using pyridine as solvent. The 0.8 mL pyridine and 250 ^L BSTFA were added to 5 mg extract and the mixture was heated at 100°C for 15 min for derivatization reaction. Oven temperature used to analyze silylated samples was as follows: 10 min at 50°C; from 50°C to 150°C with 4°C/min heating rate and then, from 150°C to 280°C with 4°C/min rate and maintained at this temperature for 15 min. Inlet temperature was 220°C. Solvent delay was 18 min. The ionization energy was 70 eV and mass range was 50—450 amu. Duplicate samples (1 ^L) were injected into GC-MS with 1 : 10 split ratio. The oven program for nonsilylated samples in diethyl ether solvent was as follows: 5 min at 40°C; from 40°C to 150°C with 4°C/min heating rate and maintained at this temperature for 5 min and then heating from 150°C to 300°C with 4°C/min rate and maintained at this temperature for 15 min. Solvent delay was shorter than that ofused for silylated samples to be able to determine more volatile compounds in the extracts (6 min). The composition of the samples were identified by NIST 2002 (verison 2.0) mass spectral library.
HPLC analysis. Rosemary extracts were analyzed by a LC-6AD Shimadzu high performance liquid chromatograph equipped with SIL-10AF Shimadzu auto injector (Shimadzu, Kyoto, Japan) and SPD-M10A VP diode array detector (DAD). Varian Microsorb-MV 100-5 C18 (250 x 4.6 mm, 5^) column (Virian, Walnut Creek, CA) were the stationary phase. The mobile phase of 2.5% of formic acid and methanol at a flow rate of 0.8 mL/min was used with a gradient program as described by Shan et al., 2005 [17]. The injection volume was 20 ^L and duplicate analyses were performed for all samples.
Determination of total phenolics content. The total polyphenolic contents of the extracts were assayed by the Folin-Ciocalteau assay [18, 19] with slight modifications. The 0.2 mL et
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