Comparative study of phytochemical profiles and morphological properties of some Damask roses from Iran

Rosa damascena is an aromatic rose species, which is cultivated for its essential oil, and is widely used in perfume, cosmetic, pharmaceutical, and food industries in the world. This experiment was conducted to evaluate essential oil and morphological variations of 26 Damask rose genotypes. For this purpose, the effect of harvest time, i.e., early morning or evening, and sampling type, i.e., fresh or dried petals, on oil content was evaluated. In addition, the composition of essential oil of the genotypes was determined using gas chromatography–mass spectrometry (GC–MS). Results showed that early morning was the preferable time for flower collection based on oil content. Furthermore, the oil yield of fresh petals was higher than that of the dried petals. Twenty-five volatile compounds were found in the extracted oils. β-Damascenone, a key marker for the quality of rose oil, was found in 22 genotypes and was more than 1.5% concentration in G3, G6, and G11 genotypes. The highest components of the oil of Damask rose genotypes were nonadecane (42.51%), β-citronellol (40.82%), n-heneicosane (34.69%), geraniol (27.76%), and n-tricosane (14.2%). A wide variation in flower characteristics, such as petal color (from white to nearly red) and petal numbers from about 25 to 95, were also recorded. The G2, G5, and G15 genotypes, originated from Isfahan, Fars, and Kerman, respectively, were selected based on petal number, flower weight, and essential oil content in fresh and dried petals. Results suggest that morphological and biochemical diversity of Damask rose genotypes can be used effectively to characterize genetic diversity between different genotypes and to select special traits in breeding programs.


Background
Damask rose (Rosa damascena Mill.) is a supreme fragrance species in the Rosaceae. It is derived from Rosa gallica and Rosa moschata [1]. This species is cultivated for its ornamental value and also for essential oil extraction in most parts of the northern hemisphere [2,3]. Iran has been introduced as the genetic diversity center and the origin of Damask roses [4][5][6][7][8][9][10]. Nowadays, this species is cultivated extensively in Bulgaria, Iran, Turkey, France, Italy, Morocco, the USA, and India [11]. The global production of rose oil is about 4.5 tones per year [12]. The global rose oil market was valued at 278.7 million USD in 2018 [13]. Products of Damask rose, including essential oil, rose water, rose concrete, dried petals, dried flower buds, and rose absolute, are used in perfume, cosmetic, pharmaceutical, and food industries. Several pharmacological attributes, such as antibacterial, antioxidant, and anti-HIV effects have been found in rose oil [14][15][16].
Rosa damascena is cultivated in widespread ecological conditions, but specific climatic conditions are needed to produce high-quality essential oil. The quality of essential oil and flower yield of R. damascena is mainly affected by geographical origin and climatic conditions, time and stage of flower harvesting, method of extraction [5], and agricultural practices [17][18][19][20][21]. Rosa damascena grows wild in some parts of Iran and has vegetatively been propagated and long been cultivated [6]. Thus, various cultivars of Damask rose have been selected during the long cultivation history, and it has also been crossed naturally with local rose species [22]. The phenotypic homogeneity caused by continuous vegetative reproduction and environmental effects makes its mass production possible to produce rose oil [5,9,23].
The most important compounds of rose oil are β-citronellol, nonadecane, geraniol, eugenol, heneicosane, and phenols such as eugenol [24,25]. In addition, some factors such as the concentration of ethanol used for extraction, storage period, and production conditions of flowers, can also affect key compounds in rose oil [5]. Several studies have been conducted on the chemical composition of essential oil in various populations of Damask rose by GC/MS through different extraction methods [3,9,15,[26][27][28], and also on genetic and morphological diversity of Rosa damascena [6,10,[29][30][31][32]. The effect of micro-climate on Damask rose cultivation and the oil composition has been also reported [32,33].
As we have access to the wide genetic diversity of Rosa damascena in Iran, it will be valuable to characterize their specific morphology and biochemical characteristics in more detail. Thus, the present study was carried out to determine the variations in the flower yield and morphological and chemical compositions of 26 different Iranian Damask rose genotypes by using gas chromatography-mass spectrometry (GC-MS).

Plant materials and collection site
26 Damask rose genotypes were selected for this study. These genotypes were previously collected from several parts of Iran (Table 1) and established in the research station for the Department of Horticulture, University of Tehran, Karaj, Iran (latitude 35°0.77ʹ N, longitude 50°0.93ʹ E and altitude 1251 m), based on a randomized block design with three replications in 2004 [8]. All samples were the same in size because of their yearly pruning. The average plant high was 165 cm and plant diameter was 123 cm. The current experiment was carried out during 2016-2018.

Evaluation of plant vegetative and flower characteristics
Morphological characteristics, such as plant height, crown diameter, number of main stems in each plant, number of flowers in main stems, angle of the secondary branches, internode length, and thorn density (one to five from high to low), were determined in 12 years old plants. Additionally, the length of stipule, peduncle, receptacle, and flower bud length and diameter prior to the opening stage were measured. Petal colors were determined visually and also measured with a colorimeter (Minolta CR-400 Chroma meter, Konica Minolta Sensing, Inc., Osaka, Japan) using the following parameters: L* (lightness), a* (redness), and b* (yellowness). Color parameters were obtained through reflectance values and chroma calculated by the following formula [34]:

Isolation and content of essential oil
For the extraction of essential oil, fresh flowers from each accession were randomly collected (20 fresh flowers per accession). Flowers were harvested both in the morning and evening. For dried samples, the collected flowers were spread on wire shelves and kept in the shade for 2 weeks at room temperature [35]. A total of 200 g of fresh petals and 55 g of dry petals (equivalent to 200 g fresh petals) were subjected to hydrodistillation using 400 ml distilled water in a clevenger for 3 h with three replications [64]. The essential oil was measured directly in the extraction burette and the oil content (v/w) in flower was expressed as percentage on a fresh weight basis of essential oil per 200 g of fresh petals. The extracted oils were transferred into vials and stored at 4°C in the dark.

Gas chromatography (GC-FID) and (GC-MS)
GC-MS analysis of the oil samples was performed on a Thermo-UFM (Ultra-Fast model, Italy) gas chromatograph equipped with a P5 (non-polar) capillary column (10 m × 0.1 mm), which employed helium (0.5 ml/min) as the carrier gas to split injection at 1:100. The oven temperature was set at 60 °C for 30 min, FID detector temperature was programmed at 285 °C at the rate of 80 °C/min, and the injector temperature was 280 °C. The relative amounts of individual components were calculated based on the GC peak areas by using a normalization method regarding response factor. The essential oil constituents were identified following an injection of n-alkanes (C 8 -C 24 ) under the same conditions and confirmed according to Wiley 275-L library and literature [36][37][38]. The compounds were identified using commercial mass spectral libraries (NIST 05, Wiley 7th Mass spectra register) [37].

Statistical analysis
For the evaluation of morphological characteristics of vegetative and flower parts, the experiment was arranged in a randomized complete block design (RCBD) with three replications. Mean values were compared at 95% (p ≤ 0.05) and 99% (p ≤ 0.01) confidence intervals using the LSD test by Minitab 16 [39].

Oil content in fresh and dry petals
In the majority of selected Damask rose genotypes, petals harvested in the morning time for dried and fresh petals had higher oil content in comparison with samples collected in the evening time with the exception of G9, G11, and G23 genotypes, for which a opposite trend was found (Fig. 1). The highest oil content in fresh petals was found in G21 for morning and evening harvest time, 0.14 and 0.15 (v/w%), respectively. Additionally, in G18, G19, G20, G24, and G25 genotypes the total volume of essential oil content in both harvesting times was similar (Fig. 1). In dried petals, the time of harvesting also affected the oil content. However, the oil content in dried petals was generally lower than in the fresh petals. Although in most genotypes a higher oil content of dried petals was recorded for the morning harvest time, there was no difference in the content of essential oil of dry petals between harvest times in G1, G2, and G4 genotypes. However, due to later flowering of G22 (0.05 v/w%) and G24 (0.04 v/w%) genotypes, the oil content in dried petals harvested in the evening time was more than in those harvested in the morning time (Fig. 2).

Morphological traits
There were clear differences in morphological characteristics between selected Damask rose genotypes ( Table 2) Table 3). Thorn density was negatively correlated with the flower bud length (r = − 0.80) and peduncle length (r = − 0.78). Moreover, a positive correlation (r =− 0.60) was found between number of nodes in the branch and flower bud length. A significant (P = 0.01) positive (r = 0.74) correlation was found between flower bud length and peduncle length ( Table 3). The flower peduncle length was positively correlated with most traits evaluated in this study. Different petal colors, from white to dark purple, were observed in the selected Damask rose genotypes ( Table 1). The measurements of color parameters gave different values of L*, a*, and b*. The results showed highly significant differences among genotypes for all color traits (Table 4). Chroma values were also different between genotypes.
In the current study, neral was present in all genotypes, except in G3, G11, G14, and G18. A major concentration of neral was in G9 (10.83%) and G2 (10.25%). Geranial was found in G1, G2, G4, G5, G9, G10, G15, and G16 genotypes at low levels ( Table 5). Neral and geranial are citral isomers, which have been found in Damask rose essential oil [28]. Farnesol, natural sesquiterpene alcohol in essential oils, was found to have the potential for alleviating massive inflammation, oxidative stress, and lung injury [41,42]. Farnesol has been widely used in cosmetics, pharmaceuticals, industrial materials, and as a material for carotenoid and tocopherol [43]. Farnesol is a sesquiterpene trans and exists in some Damask genotypes. A higher amount of it was found in G17 (3.01%), and the highest e-e Farnesol was observed in G15 (8.28%).
Rose oxide is an insignificant component of rose oil [44]. In this study, the rose oxide has been found at low concentrations in G3, G11, G15, G19, G22, G23, and G26 (Table 5). Phenethyl alcohol is an enjoyable floral perfume belonging to aromatic alcohols, and one of the main components of rose hydrosols, which is mainly used in perfumery [2]. However, this compound was detected at low levels only in some genotypes including G6 (1.54%), G23 (0.40%), and G2 (0.33%). Phytol is a major component of plant-derived essential oils. It has been recognized for its wide range of pharmacological effects on the nervous system, including anxiolytic, antidepressant, and antimicrobial [45][46][47]. Several recent studies have suggested that some phytol-derivatives (phytanol, phytanyl amine, and phytanyl mannose) target tumor cells by induction of the expression of a range of chemokines and cytokines effects [48,49]. Other hydrocarbon-like ingredients, n-docosane and n-tricosane, were also identified in Damask roses essential oil. Quantities of n-tricosane were much more than that of n-docosane in all genotypes. In the present study, G6 (9.33%) and G20 (14.20%) genotypes showed the highest contents of n-tricosane and n-docosane, respectively.

Discussion
Several studies have been conducted to date on the genetic diversity of R. damascena in Iran, which have shown a high diversity and genetic variation of this species [6,8,50]. In this study, R. damascena genotypes  showed a remarkable diversity in petal color from dark pink (G3/Tehran genotype) to pale pink (G9/Fars genotype) and white (G2/ Isfahan and G26/ East Azerbaijan genotypes). However, the majority of them were pink or pinkish ( Fig. 3; Table 4). Some anthocyanins such as pelargonidin and cyanidin in the petal cells are responsible for the color of rose flowers [51]. Petals of industrial oilbearing damask roses grown in the world are typically pink, while wild roses usually have pink or white flowers [52]. Karami et al. [33] reported a positive relationship between essential oil content and anthocyanin concentration in Damask rose.
The number of petals is a very important indicator of the total essential oil. Significant negative correlations between thorn density and morphological characteristics, excluding bud diameter, were observed. Additionally, there was a significant positive correlation (0.39**) between the number of petals and thorn density (Table 3). Therefore, it is possible to select genotypes with a higher flower weight and number of flowers in attempts to improve the flower yield and essential oil content [7,32].
According to the results (Figs. 1, 2), harvesting time had a major effect on essential oil content, and the morning harvested flowers had a higher essential oil content. Moreover, there was no positive relationship between oil content and petal number. This is consistent with the results of some reports, in which the oil content of the damask rose flowers depended on the time of harvesting, and the petals harvested in the morning had a higher oil content [5,23]. Results of the current study also showed that the essential oil content was influenced by harvesting time in the majority of 26 genotypes of the Damask rose, confirming that morning time was the optimal time for harvest, which is consistent to earlier reports [53][54][55][56]. Large differences in the content of essential oils (Table 5) were observed between 26 selected Damask rose genotypes, which is in agreement with the results of researches who reported high variations in the volatile compounds of Damask rose oil [11,18,32]. It has been reported that the quantity and composition of essential oil ingredients are significantly influenced by the genotype and agronomic conditions, as well as plant and flower developmental stage and harvesting time [57][58][59]. Overall, the content of monoterpenes (citronellol, nerol, and E-geraniol), sesquiterpenes, and aliphatic hydrocarbons was high (Table 5). Furthermore, e-geraniol, a major rose-oil component, was high in all 26 selected Damask rose genotypes. The percentage of four major hydrocarbons (heptadecane, nonadecane, eicosane, and heneicosane) were also high in the extracted essential oils (Table 5). Similar to other reports, this study revealed high variations between Rosa damascena genotypes regarding oil content and components, morphological diversity, and petal color [9,[60][61][62][63].

Conclusions
Results from this study revealed that Damask rose genotypes in Iran have significant diversity in morphological characteristics, oil content, and also composition. The harvesting time of Damask rose flowers significantly affected the essential oil yield, and, for most genotypes, harvesting is recommended to be performed in the morning, but for higher oil content of G2 and G5 genotypes, evening harvesting time might be recommended. The varied deviations in petal colors, petal numbers, and essential oil content in genotypes were observed in this experiment. Thus, the existence of these characteristics and a good chemical variation shown in the profiling reveal that the studied collection of Damask rose is a good source for the selection of the industrial oil-bearing damask rose cultivars and those that could be used as an ornamental plant in the landscape because of its uniquely fragrant flowers. Compared with the other genotypes, G5 and G21 had the highest essential oil content. 25 volatile compounds were identified in the essential oil of Damask rose genotypes. The highest concentration of geraniol, β-citronellol, nonadecane, and β-damascenone were found in G12 (42.51%), G26 (40.82%), G5 (27.76%), and G3 (1.76%) genotypes, respectively. It has been found that the most abundant compounds are of several main classes including alcohols (citronellol, geraniol, nerol) and hydrocarbons (heptadecane, nonadecane, eicosane, and heneicosane). In conclusion, the morphological and biochemical diversity of Damask rose genotypes can be used effectively to characterize genetic diversity between different genotypes and to select special traits in breeding programs.