Supercritical CO2 Extraction of Narcissus poeticus L. Flowers for the Isolation of Volatile Fragrance Compounds

The flowers of Narcissus poeticus are used for the isolation of valuable fragrance substances. So far, as the majority of these substances consist of volatile and sensitive to heat compounds, there is a need of developing effective methods for their recovery. In this study, freeze-dried N. poeticus inflorescences were extracted with pure supercritical CO2 (SFE-CO2) and its mixture with 5% co-solvent ethanol (EtOH) at 40 °C. Extract yields varied from 1.63% (12 MPa) to 3.12% (48 MPa, 5% EtOH). In total, 116 volatile compounds were identified by GC-TOF/MS in the extracts, which were divided into 20 different groups. Benzyl benzoate (9.44–10.22%), benzyl linoleate (1.72–2.17%) and benzyl alcohol (0.18–1.00%) were the major volatiles among aromatic compounds. The amount of the recovered benzyl benzoate in N. poeticus SFE-CO2 extracts varied from 58.98 ± 2.61 (24 MPa) to 91.52 ± 1.36 (48 MPa) mg/kg plant dry weight (pdw). α-Terpineol dominated among oxygenated monoterpenes (1.08–3.42%); its yield was from 9.25 ± 0.63 (12 MPa) to 29.88 ± 1.25 (48 MPa/EtOH) mg/kg pdw. Limonene was the major monoterpene hydrocarbon; (3E)-hexenol and heneicosanol dominated among alcohols and phenols; dihydroactinidiolide and 4,8,12,16-tetramethyl heptadecan-4-olide were the most abundant lactones; heptanal, nonanal, (2E,4E)-decadienal and octadecanal were the most abundant aldehydes. The most important prenol lipids were triterpenoid squalene, from 0.86 ± 0.10 (24 MPa) to 7.73 ± 0.18 (48 MPa/EtOH) mg/kg pdw and D-α-tocopherol, from 1.20 ± 0.04 (12 MPa) to 15.39 ± 0.31 (48 MPa/EtOH) mg/kg pdw. Aliphatic hydrocarbons (waxes) constituted the main part (41.47 to 54.93%) in the extracts; while in case of a 5% EtOH the percentage of alkanes was the lowest. The fraction of waxes may be removed for the separation of higher value fragrance materials. In general, the results obtained are promising for a wider application of SFE-CO2 for the recovery of fragrance substances from N. poeticus flowers.


Introduction
Aromatic herbs and flowers are an important source of natural ingredients for the development of flavours and fragrances. Narcissus is a genus of perennial spring flowering plants of the Monocotyledon family Amaryllidaceae. Carl Linnaeus originally defined six species of Narcissus in 1753, while N. poeticus was the first one described in his book Species Plantarum [1]. Nowadays the genus is generally considered to consist of about 12 sections with approximately 80 species. Poetic, commonly also called daffodil (N. poeticus), supposed (N. pseudonarcissus), tripod (N. triandrus), and bouquet (N. tazetta) narcissus are the most widely grown species, while cyclamen (N. cyclamineus), chaste (N. papyraceus), and fragrant (Narcissus × odorus) are less popular. Three species, namely white (N. poeticus), bouquet (N. tazetta) and yellow/supposed (N. pseudonarcissus) are most popular in Lithuania as ornamental plants in flower gardens, which also grow in old parks and meadows.
N. poeticus is a wild, bulbous herbaceous plant, growing up to 20-40 cm height. The leaves are radical, green, narrow and long; it blooms in late spring (April-May) with an extremely fragrant white flower per stem [2]. N. poeticus is thought to originate from the Volatile compounds of N. poeticus have also been studied [2,11,21,22]. Ehret et al. [22] identified 80 new minor volatile components in N. poeticus absolute from France "Massif central", α-terpineol (23.7%), methyl (E)-isoeugenol (20.0%), and benzyl benzoate (19.4%) being the major ones; more than 20 of them were considered as responsible for the complex floral notes reminiscent of "rose", "jasmine", "violet", "tuberose" and "orange flower". Cinnamyl alcohol (29.91 µg/g), methyl isoeugenol (28.07 µg/g), isoeugenol (23.12 µg/g), methyl eugenol (20.72 µg/g), and α-terpineol (20.31 µg/g) were the main components in hexane extract of fresh N. poeticus flowers of Rocca di Mezzo [11], whereas cinnamyl alcohol (30.2 µg/g), benzyl benzoate (28.5 µg/g), isoeugenol methyl ether (28.1 µg/g), (Z)ocimene (25.2 µg/g), isoeugenol (22.9 µg/g) were quantitatively major in the plants from Sirente-Velino (Abruzzo region, Italy) [2]. To the best of our knowledge, only one article is available on SFE-CO 2 of N. poeticus [11]; however, this study applied comparatively low pressure of the fluid CO 2 (12 MPa). So far, as the solubility of many compounds increases with increasing solvent density, which depends on its pressure, in our study we applied CO 2 pressure up to 48 MPa. It is important to mention that some of these volatiles are stated as more or less allergenic compounds, for example, cinnamyl alcohol, isoeugenol and cinnamyl aldehyde, and their use must follow legislation. It was suggested that in the preparation of safer perfumes absolute can be obtained from the corona only instead of tepals plus corona [2].
Considering previously reported data, our study aimed at evaluating the effects of pressure and addition of co-solvent in SFE-CO 2 of freeze-dried N. poeticus flowers. Extract yields and comprehensive analysis of the recovered volatiles compounds were performed for this purpose. It is expected that the data obtained will expand our knowledge on the composition of N. poeticus fragrant constituents and the possibilities of their recovery by using green extraction techniques.

Extract Yields
The yields of the SFE-CO 2 extracts of N. poeticus freeze-dried flowers varied from 1.63 ± 0.29 to 3.12 ± 0.12% (Table 1). The temperature of 40 • C, static extraction time 10 min and dynamic extraction time of 120 min were kept constant in all experiments. According to the literature reports, the yields of the concrete (absolute + waxes) obtained with hexane extraction were 0.2-0.3% [22], 0.41-0.45% [2,11] of fresh flowers and with petroleum ether -0.2% of fresh flowers [23]. The percentages of absolute in the concretes varied from 27 to 37% [11,23]. Table 1. SFE-CO 2 extraction conditions of Narcissus poeticus and obtained yields (%).

SFE-CO 2 Extraction Conditions
Extract Yield, % There were no statistical differences in the extract yields at 12-36 MPa, while at 48 MPa the yield of the recovered fraction was significantly higher (p < 0.05). Additionally, 5% ethanol applied as a co-solvent increased extract yield by~32% (from 2.36 to 3.12%) compared with pure SFE-CO 2 at 48 MPa. Ferri et al. [11] compared conventional hexane extraction with SFE-CO 2 of N. poeticus; however, the absolute yields of SFE-CO 2 extracts are not available in their article. Nevertheless, it is evident that SFE-CO 2 extracts contained all the main constituents, which were identified in hexane extracts, while their yields in SFE-CO 2 were significantly lower, even in case of using 5% ethanol, which enhanced the yields of most characteristics compounds [11].
Benzyl benzoate, benzyl alcohol and benzoic acid are used in a wide variety of cosmetics formulations as fragrance ingredients and preservatives. The group of benzyl derivatives was reaffirmed as generally recognized as safe (GRAS) by the Expert Panel of the Flavour and Extract Manufacturers (FEMA), and the evidence of safety is supported by the fact that the intake of benzyl derivatives as natural components of traditional foods is larger than their intake in the case of intentional adding as flavouring substances [24]. Benzyl alcohol is an aromatic alcohol, which has been characterised as possessing "sweet, flower" [25] and "berry, cherry, grapefruit, citrus, and walnut" [26] odour notes. The absolute amounts of main volatile compounds in mg/kg pdw are summarised in Figures 1 and 2. The highest recoveries of benzyl alcohol (12,8.79 ± 0.59 mg/kg pdw), as well as benzyl linoleate (107, 18.96 ± 0.42 mg kg pdw) were obtained at 48 MPa/EtOH; however this value was not significantly different to the one obtained at 48 MPa ( Figure 1A). Benzyl benzoate and benzyl alcohol were previously reported in high amounts in the absolutes of hexane extracts of N. poeticus from Italy [2,11].         Table 2. Values within columns followed by the same letter (a-e) do not differ statistically at p < 0.05 (Duncan test).  Table 2. Values within columns followed by the same letter (a-e) do not differ statistically at p < 0.05 (Duncan test).
It was reported that narcissus absolute has "a very strong, green, earthy and woody" odour, and that in appropriate dilution, releases a characteristic blend of floral and balsamic notes: light floral notes (rose or jasmine), deep floral notes (ylang-ylang, tuberose, orris and violet), notes of the balsamic type (styrax) and woody, earthy scents (oakmoss or patchouli) [22]. The balsamic type (styrax) flavour notes can be associated with the presence of benzoates and cinnamates in the N. poeticus SFE-CO 2 products composition. Benzyl benzoate also possesses "balsamic, oil, herb" [25] and "almond, cheese, cherry, floral, pineapple, strawberry, sweet" [26] aroma notes. The absolute amount of the recovered benzyl benzoate from N. poeticus by SFE-CO 2 varied from 58.98 ± 2.61 mg/kg pdw (24 MPa) to 91.52 ± 1.36 mg/kg pdw (48 MPa) ( Figure 1A).
It was also reported that benzyl benzoate, its derivatives and benzyl cinnamate may be promising compounds in reducing hypertension [28]. It may be concluded that N. poeticus SFE-CO 2 extracts after the separation of waxes might be a potential natural source of benzyl benzoate and benzyl alcohol.
Limonene was the major monoterpene hydrocarbon; however, its percentage was low (0.08-0.17%). It is interesting that (E)-β-ocimene was not found in SFE-CO 2 extracts of N. poeticus from Lithuania, while this compound was reported to be present in six species of Narcissus from Spain [19], N. tazetta EOs from Greece and N. poeticus absolute from Italy [2].
The recoveries of esters ( Figure 1D) and acids ( Figure 2A) were quite low; however, it is interesting to mention that the amounts extracted from N. poeticus ethyl hexadecanoate (78), ethyl linolenate (90) and linoleic acid (86) significantly increased at 48 MPa and adding 5% of EtOH. It is believed that high amount of alcohols and long-chain acids in narcissus has more effect on the longer lasting trait of its odour, rather than on the odour quality [17].

Triterpenoids, Tocopherols and Others
Supercritical CO 2 together with volatile compounds also extracts higher molecular weight lipophilic compounds, among them important prenol lipids such as triterpenoid squalene and tocopherols, which are well known antioxidants. Squalene is a long chain triterpene hydrocarbon, which is a precursor in the synthesis of sterols. The amount of squalene varied from 0.86 ± 0.10 mg/kg pdw (24 MPa) to 7.73 ± 0.18 mg/kg pdw (48 MPa/EtOH). It is worth noting that the use of a co-solvent ethanol increased the amount of extracted squalene~2.5 times ( Figure 2D). The antioxidant and oxygen carrying properties of squalene predicts its potential use in preventing cardiovascular diseases and cancer [34].
The amount of the recovered vitamin E (D-α-tocopherol) was from 1.20 ± 0.04 mg/kg pdw (12 MPa) to 15.39 ± 0.31 mg/kg pdw (48 MPa/EtOH). In nature, there are four main structures of tocopherols, namely α-, β-, γ-, and δ-tocopherol. Tocopherols are lipophilic molecules which are synthesized by plant cells and stored in leaves and seeds, and are endowed with antioxidant functions. They possess multiple beneficial healthy effects, such as the prevention of cardiovascular diseases and cancer [35].
The highest absolute amounts of aliphatic hydrocarbons were extracted at 12 MPa, while the lowest at 24 MPa. Further increase in pressure from 24 to 48 MPa resulted in the increase in the recovered amounts of n-alkanes, while in the case of using ethanol it was reduced again. For example, the highest amount of the recovered n-heptacosane (105) was 131.49 ± 7.00 mg/kg pdw at 12 MPa and the lowest 84.07 ± 1.37 mg/kg pdw at 24 MPa (Figure 3). At further increase in pressure its amount increased to 121.88 ± 5.30 mg/kg pdw (48 MPa), while 5% ethanol reduced the value to 98.08 ± 5.30 mg/kg pdw, which was significantly lower to that extracted at 12 MPa. The amount of these saturated acyclic alkanes could be reduced by using conventional dewaxing procedures.  Table 2. Values within columns followed by the same letter (a-e) do not differ statistically at p < 0.05 (Duncan test).

Plant Material and Chemicals
Narcissus poeticus L. plants were grown in a farmstead near the city of Klaipėda (Lithuania, coordinates: 55 • 45 N 21 • 10 E) and the flowers were collected at a full flowering stage. The flowers were picked in the morning manually, separated from the stems by scissors. Fresh flowers were frozen to −40 • C and freeze-dried in a Sublimator 40 at 0.05 mbar pressure (Zirbus Technology, Bad Grund, Germany). The dried flowers were ground in an ultra-centrifugal mill ZM 200 (Retsch, Haan, Germany) using 0.5 mm hole size sieve.

Supercritical Carbon Dioxide Extraction (SFE-CO 2 )
SFE-CO 2 was carried out in a supercritical fluid extractor Helix (Applied Separation, Allentown, PA, USA). Each extraction was performed from 10 g of ground N. poeticus flowers placed in a 50 mL cylindrical extractor vessel (14 mm × 320 mm; h/d = 22.86), between two layers of defatted cotton wool in both ends, to avoid particles clogging the system. The temperature of the extraction vessel was controlled by a surrounding heating jacket. The flow rate of CO 2 in the system (v) was controlled manually by the micrometering valve (back-pressure regulator). The volume of CO 2 consumed was measured by a ball float rotameter and a digital mass flow meter in liters per min (SL/min) at standard state: pressure P = 100 kPa, temperature T = 20 • C, density ρ = 0.0018 g/mL. The conditions for extraction were set as follows (see in Table 1): extraction time 120 min, pressure 12-48 MPa, extraction temperature 40 • C, flow rate of CO 2 2 L/min. A static time of 10 min was included in to the total extraction time and was constant. The extraction at 48 Mpa was also performed using 5% (v/v) ethanol as a co-solvent in order to enhance the polarity of the solvent mixture. The extracts were collected in brown glass bottles at room temperature and atmospheric pressure and stored at −20 • C until further analysis. The primary oven was programmed as follows: 50 • C ramped to 100 • C at 10 • C/min (0 min) further ramped to 300 • C at 5 • C/min (hold 5 min); the secondary oven programming was from 65 • C ramped to 115 • C at 10 • C/min (0 min) then ramped to 315 • C at 5 • C/min (hold 5 min). The transfer line temperature was 250 • C, the GC injector port was kept at 280 • C with desorption time of 5 min.
The TOF/MS acquisition rate was 10 spectra/s, the mass range used for identification was from 35-550 m/z units. Detector's voltage was set at 1550 V and ion source temperature of 250 • C. Data from the GC × GC-TOF/MS system were collected by ChromaTOF software v.4.22 (LECO) after a solvent peak delay of 420 s in a splitless mode for 30 s, a further valve was opened and purge flow was 20 mL/min; mass spectrum assignment was based on matching against Adams, NIST, MainLib, RepLib mass spectra libraries; signal-to-noise threshold was set as 50 with the minimum similarity accepted was 700. The mean values were calculated from triplicate injections. Quantitative data were obtained by peak area normalization without using correction factors as the means of triplicate GC-TOF/MS runs and expressed as peak area percentage and recalculated in mg/kg per plant dry weight (mg/kg pdw). Quantitative data were calculated using the formula: m i = (m D × A i /A D × RF i ) × ((Y e × 1000/100)/m e ), where m i is the mass of the individual compound i to be quantified, expressed in mg/kg per plant dry weight (mg/kg pdw); m D mass of decane (internal standard, IS), A i and A D -are the peak area of the analyte i and the IS, respectively, RF i -response factor of individual compound; Y e -the yield of the extract (%), m e -the mass of the extract (g).
The identification of volatile components was assigned by comparing their Kováts Retention Indices (KI) relative to C 7 -C 30 n-alkanes, obtained on nonpolar BPX-5 column with those provided in literature [27] and by comparing their mass spectra with the data provided by the NIST, MainLib, RepLib and Adams mass spectral libraries. The identity of some constituents was confirmed by co-injection of reference compounds. Positive identification was assumed when good match of mass spectrum and KI was achieved.

Statistical Analysis
All analyses were replicated at least three times and all data are reported as mean values ± standard deviations (SD) using MS Excel 2010 software. Data were statistically handled by one-way analysis of variance (ANOVA, vers. 2.2), significant differences among the samples were evaluated by the Duncan's multiple-range test at the probability level of p < 0.05.

Conclusions
Narcissus poeticus flowers were extracted with supercritical carbon dioxide at different solvent pressures, from 12 to 48 MPa. The yield of lipophilic fraction significantly increased after raising the pressure from 36 to 48 MPa and by adding 5% of a co-solvent ethanol into the CO 2 flow. Generally, the yields were higher compared with the previously reported data for conventional extraction with organic solvents. In total, 116 volatile compounds were identified by GC-TOF/MS in the extracts. The most important for N. poeticus odour constituents benzyl benzoate (9.44-10.22%), benzyl linoleate (1.72-2.17%) and benzyl alcohol (0.18-1.00%) were the major volatiles among aromatic compounds. The extracts contained a large fraction of waxes, which are not desirable in the production of higher quality fragrance ingredients; however, adding a co-solvents ethanol enabled the reduction in the percentage of higher alkanes, while the amount of the recovered benzyl aromatics increased. On the other hand, for practical purposes, SFE-CO 2 at 48 MPa would most likely be preferable because in this case the process becomes less complicated and cheaper; there is no need for a co-solvent pump and removal of it after extraction, which may result in some loss of volatile fragrance constituents. However, adding co-solvent increases the recovery of an important bioactive compound α-tocopherol approx. 2-fold. In general, the results obtained are promising for a wider application of supercritical extraction for the recovery of fragrance substances from Narcissus poeticus flowers.