Introduction
Aromatic and volatile products of plant secondary metabolism are used in the pharmaceutical, chemical, cosmetic, and food industries (Gourine et al., 2010; Hosni 2011; Ginova et al., 2012). In recent years, there has been an increasing interest in the use of natural substances due to concerns about the safety of some synthetic compounds, which have encouraged more detailed studies on originated substances.
Damask rose is one of the most important aromatic plants in the world. It is cultivated in Iran, Turkey, Bulgaria, India, South France, China, South Italy, Libya, South Russia, and the Ukraine (Ginova et al., 2012). Recently, medicinal properties such as anti-HIV, antibacterial, antiseptic, antioxidant, antiviral, aphrodisiac, antitussive, hypnotic and, antidiabetic effects, relaxant effects on tracheal chains and a tonic for the heart, liver, stomach and uterus have been reported for this plant (Boskabady et al., 2011).
The Damask rose flowers once in a year in temperate climates at the beginning of summer and twice
in tropical climates. The blooming period is extremely short (35-40 d) with low content of aromatic, and volatile products (Najem et al., 2011; Karami et al., 2012; Kumar et al., 2013a). The essential oil content of flowers distilled after 36 h of storage was only 40% that of fresh flowers (Baydar and Gokturk Baydar 2005). Storage of flowers at low temperatures in airtight plastic envelops retained their aromatic, and volatile content (Kumar et al., 2013b). Baydar et al. (2008b) reported that the fermentation of rose flowers increased the levels of citronellol and methyl eugenol while reducing the levels of geraniol and nerol. Kumar et al. (2013b) have reported that storing rose flowers at 4°C for up to 96 h does not affect their essential oil content. During peak flowering when there is a flower glut, flowers cannot be distilled promptly due to either a shortage of distillation apparatus or some technical fault in the distillation unit, resulting in a loss of product quality due to fermentation of flowers. The cold storage of precious flowers can minimize or even overcome this problem. Thus, the present study was conducted to investigate the effect of post-harvesting storage conditions on the composition of Damask rose oil in southwest of Iran.
Materials and Methods
To determine the best post-harvesting methods to dry the R. damascena Mill., a randomized complete design was conducted in April-July 2016-2017 with three replications in Shahrekord region of Chaharmahal and Bakhtiari province southwest of Iran. The soil and climatic properties of the sampling zones are listed in Tables 1 and 2. This research was done to study the effects of temperature [namely, room temperature (25°C), refrigerator temperature (4°C), and freezing temperature (-20°C)] and the time of storage of flowers (6, 12, 24, 48 and 96 h) on the content and composition of the essential oils of R. damascena. Control treatment was 6 h and 25°C.
Essential oil content was determined by distilling flowers in Clevenger apparatus. 1000 g of rose flowers was placed in 6 L Clevenger type distillation apparatus and distilled for 5 h with 3 L of pure water. The quantities of rose oils obtained at the end of distillation were measured as mL and % ratios (w/w) were determined by multiplying the oil content with oil density i.e., 0.858. The unit of the oil content is g/100 g (fresh weight). All the essential oil samples were dried over hydrous sodium sulphate and stored at 4°C until analyzed by GC and GC-MS analysis. Voucher specimens (20074-TUH) of the species have been deposited in the Herbarium of the Center of Agricultural and Natural Resources of Chaharmahal and Bakhtiari Province, Shahrekord, Iran. Plant species were identified by Mozaffarian (2008).
Ground GC analysis was done on an Agilent Technologies 7890 GC equipped with FID and a HP-5MS 5% capillary column. The carrier gas was helium at a flow of 0.8 mL·min-1. Initial column temperature was 60°C and programmed to increase at 4°C/min to 280°C. The split ratio was 40:1. The injector temperature was set at 300°C. The purity of helium gas was 99.99% and 0.1 mL samples were injected manually in the split mode. GC-MS analyses were carried out on a Thermo Finnigan Trace 2000 GC/MS system equipped with a HP-5MS capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm). Oven temperature was held at 120°C for 5 min and then programmed to reach 280°C at a rate of 10°C/min. Detector temperature was 260°C and injector temperature was 260°C.
The compositions of the essential oil were identified by comparison of their retention indices relative to a series of n-alkanes (C7-C24), retention times and mass spectra with those of authentic samples in Wiley library (Adams, 2007; Shamspur and Mostafavi, 2010).
All data were subjected to ANOVA and simple pearson correlation indices using the statistical computer package SAS and treatment means separated using LSD’s multiple range test at p < 0.05 level.
Results and Discussion
Storage of flowers under different conditions affected their essential oil content and composition (Table 3). There were significant differences in correlations between components (Table 4). In total, 39 components (Table 5, 6) of R. damascena essential oil were identified and quantified using GC and GC-MS in the present study. These compounds constituted 97.2 ± 4.8% to 97.82 ± 3.6% of the oil. It was reported that monoterpene alcohols were the main components of rose oil and that these components gave the oil its characteristic aroma. It seems that an increase in the level of each main essential oil component was associated with a decrease in the level of another component. There was a significant positive correlation between essential oil content and the main essential oil components such as beta citronellol, geraniol, trans geraniol, heptadecene and pentadecane but a negative relationship between essential oil content and other main of essential oil components such as nonadecane and heneicosane (Table 4). Essential oil content obtained from distillation of Damask rose flowers during storage is presented in Table 5 and Table 6. In the present study, the oil content decreased significantly with increasing storage duration. Maximum essential oil content was recorded at 0.054% to 0.055% in flowers stored at -20°C for 6 h while minimum essential oil content (0.011% to 0.019%) was recorded in flowers stored at 25°C for 96 h. This decrease in the essential oil content had due to an increase in fermentation during storage. Earlier, Baydar and Gokturk Baydar (2005) reported a decrease in essential oil content during storage due to fermentation. Baydar et al. (2008b) reported a 0.25% reduction in the oil content of flowers stored at 25°C for 96 h compared with flowers distilled immediately. Likewise, Kumar et al. (2013a) recorded 8.5% and 27.6% reductions in essential oil content when Damask rose flowers were stored for 24 h at 4°C and 18°C, respectively, without using any packaging material. While studying the effect of different storage temperatures (0°C and 3°C) and durations (7, 14, 21 and 28 d) on oil yield and essential oil components of R.damascena, it was found that the effect of storage temperatures on oil content was not significant but the effect of storage duration was (Kazaz et al., 2009). Temperature and humidity were found to play a vital role in the biosynthesis or accumulation of essential oils in stored herbs (Sharma and Kumar, 2016) just as temperature plays an important role in maintaining the post-harvest quality and extending the shelf-life of fruits and vegetables (Kazaz et al., 2009). In another study by Mohamadi et al. (2011) in Iran, the essential oil content of rose petals stored at -20°C was not significantly affected by storage for up to 21 d. The concentrations of the major components beta citronellol, nonadecane, geraniol, trans geraniol, heneicosane, heptadecene and pentadecane changed during storage at 25°C compared to those stored at -20°C for a similar duration.
The concentration of components with a lower molecular weight decreased with prolonged storage time. The total concentrations of the main components beta citronellol, nonadecane, geraniol, trans geraniol, heneicosane, heptadecene and pentadecane were 55 ± 2.4 (first year) to 65.72 ± 1.9 (second year) in the control treatment (25°C × 6 h). Beta citronellol, geraniol, trans geraniol, heptadecene and pentadecane concentrations increased (65.7 ± 3.1% to 65 ± 1.1%) up until 6 h of storage at -20°C but declined thereafter. This may have been due to the lower boiling points of these compounds. Rowshan et al. (2013) and Najafian (2014) also reported similar results for Thymus daenensis and Melissa officinalis L., respectively. Kazaz et al. (2009) observed an increase in the level of citronellol in rose flowers stored at 0°C and 3°C for 28 d in Turkey. The increase in levels of beta citronellol, geraniol, trans geraniol, heptadecene and pentadecane may occur at the expense of nonadecane and heneicosane and their levels improved to 20.6 ± 0.09% and 21.3 ± 0.6% at -20°C × 96 h from low levels of 9.4 ± 0.07% to 9 ± 0.5% at -20°C × 6 h. There was a decrease in the concentration of beta citronellol, geraniol, trans geraniol, heptadecene and pentadecane from 65.7 ± 3.1% to 65 ± 1.1% and 38.79 ± 1.9% to 40.34 ± 1.2% at -20°C for storage from 6 h to 96 h in the first and second seasons, respectively. The highest percentage of beta citronellol (36.3 ± 1.2% to 35.1 ± 5.5%) was obtained at -20°C × 6 h post-harvesting. Geraniol is a major component of the Damask rose that is important for perfumery and flavoring, and as per ISO 9842-2003 standards its concentration should be 15.0-22.0%. Geraniol content at -20°C decreased from 19.3 ± 0.1% to 15.1 ± 0.02% (first year) and 21.1 ± 0.1% to 15.7 ± 0.2% (second year) with an increase in storage duration from 6 to 96 h, respectively. The concentration of geraniol decreased with increases in storage duration; when its concentration fell below 15%, the essential oil content and quality decreased significantly (Kumar et al., 2013a; Sharma and Kumar, 2016). Farhath et al. (2013) reported that geraniol is a natural antioxidant that might stabilize essential oil content and quality during storage.
The total concentration of nonadecane and heneicosane increased at lower storage temperatures from 8.87 ± 2.1% (first season) and 12.71 ± 1.4% (second season) in the control group (25°C × 6 h) to 20.6 ± 1.2% (first season) and 21.3 ± 1.4% (second season) at -20°C × 96 h. Our results were component with those of Mihailova et al. (1997) in Japan for the Kazanlik rose, Kazaz et al. (2009) in Turkey and Mohamadi et al. (2011) in Iran for the Damask rose. As per ISO 9842-2003 standards, the concentration of parafins (viz., nonadecane, and heneicosane) should range between 8.0-15.0% and 3.0-5.5%, respectively. The remaining treatments recorded higher values of heneicosane, which are not desirable in the quality of rose oil. Maximum essential oil content and better quality of rose oil for perfumery industries were produced at 6 h of storage at -20°C as compared to other treatments.
Methyl eugenol (ME) is a naturally occurring compound found in many herbs and spices including the Damask rose and it is a highly valuable aromatic chemical used in cosmetic products and flavoring agents. In rose oil the maximum acceptable indicative value for ME is 3.5%, but due to its genotoxic and carcinogenic effects (Ding et al., 2011; Rusanov et al., 2012), a lower concentration of ME is desirable. Methyl eugenol concentrations in the present study were in the acceptable range regardless of storage condition (0.93 ± 0.009% to 0.08 ± 0.01% [first year)] and 0.99 ± 0.01% to 0.55 ± 0.01% [second year]). Baydar et al. (2008a, b) found that the hydrocarbon concentration in rose flowers stored for different durations at 4°C and 25°C temperature showed higher scores than flowers immediately distilled. As far as the essential oil and rose water compositions of fresh flowers of R. damascena are concerned, Verma et al. (2011) found citronellol (15.9-35.3%), geraniol (8.3-32.2%), nonadecane (4.5-16.0%), and heneicosane (2.6-7.9%) to be the major components of the essential oil. Our results are well in concordance with Baydar et al. (2008a, b), and Erbas and Baydar (2016). Grouped components were also excellent and within the desired limits produced by 6 h storage at 4°C and -20°C treatments.
The storage of Damask rose flowers at 4°C for 6 h maintained the essential oil content and quality of rose oil in accordance with international standards. Admittedly, it is better to use fresh flowers for essential oil production, but when flowers are collected in large quantities and cannot be distilled all at once in the distillation unit due to a flower glut or technical fault in the distillation unit, some parts of the flowers undergo varying degrees of fermentation before distillation. Hence, it is important to discover a method for preserving flowers before distillation. The present study revealed that it is possible to store the flowers at -20°C for 6 h without a severe loss of essential oil content and composition.
