Chemical Characterization of Rosa canina L. Rosehip Seed: Application of Raman Spectroscopy and Gas Chromatography ^†

Abstract

Rosehip seeds represent the food industry waste material, in the production of marmalade, jam, beverages, jelly, syrup, tea, etc. Agri-food wastes are rich in bioactive compounds and nutrients that can add value to different fields of agriculture and food production. The aim of this study was to assess the chemical composition of seeds from Rosa canina L. hips, with the focus on the seed oil fatty acid profile. In this respect, the analytical methods in situ Raman spectroscopy (RS) and gas chromatography (GC) were used. Fatty acids in the form of methyl esters (FAMEs) were analyzed by gas chromatography with a flame ionization detector (GC/FID). Raman spectra showed the presence of lipids, fatty acids, polyphenolics and saccharides (including cellulose) as the predominant classes of compounds in seeds. Bands at 1266, 1328, 1369 and 1655 cm⁻¹ were associated with lipids and unsaturated fatty acids (UFAs). The spectra also indicated cis isomers in the lipid fraction. Seeds contained 5.6% of oil, and GC analysis confirmed the presence of UFAs linoleic acid (ω-6) and α-linolenic acid (ω-3) (29.72 and 4.20%, respectively). Raman spectroscopy was applied as a fast and non-destructive analytical method for the chemical evaluation of rosehip seeds. The results of the GC analysis show that rosehip seeds are a good source of nutritionally valuable fatty acids that might be utilized in products specified as functional foods.

1. Introduction

Dog rose (Rosa canina L.), the well-known and traditionally used European species, has been recently considered as a complex of species (an aggregate) due to genetic and related morphological polymorphism [1]. Due to its nutritional value and sensory properties, as well as the abundance of bioactive compounds, rosehip takes a significant place in the human diet and food industry [2,3]. Rosehip fruits contain about 30–35% of seeds [4], which are considered as the waste material in the production of marmalade, jam, beverages, jelly, syrup, tea, etc. In recent years, due to their specific fatty acid composition, rosehip seeds have been used in the cosmetic and pharmaceutical industries [5]. They are a good source of linoleic, linolenic, palmitic and stearic acid. The predominant compounds are linoleic and α-linolenic acid, which are essential fatty acids that have a very important role in metabolism [6,7,8]. Fatty acids extracted from seeds also show significant antibacterial, antioxidant and anti-inflammatory activity [9].

Conventional methods for chemical composition analysis of fruits and seeds (HPLC, TLC, UV/visible spectrophotometry, etc.) usually require long procedures of standardization and involve time-consuming extraction steps and expensive chemicals [10]. On the other hand, in situ analysis by Raman spectroscopy as a rapid and non-destructive method may provide chemical and structural information with minimum requirements for sample preprocessing [11,12].

The aim of this study was to assess the chemical composition of seeds from Rosa canina L. hips using Raman spectroscopy and gas chromatography, with the focus on the seed oil fatty acid composition.

2. Material and Methods

2.1. Plant Material

About 50 rosehip (Rosa canina L.) specimens (ripened fruits) were collected from the rural area near Čačak city (locality Gornja Trnava, Moravica District, Central Serbia) in the autumn of 2018. A plant sample was deposited in the Herbarium of the Faculty of Agriculture, Belgrade-Zemun, Serbia. The collected rosehips were washed with tap water and dried at room temperature. Fleshy fruit parts (hypathium) and seeds (Figure 1a,b) were first separated and then placed at low temperature (ca. −18 °C) until the Raman spectroscopy analyses to prevent damage of the chemical composition of the samples. Additionally, for the purpose of gas chromatography analysis (Figure 1c), seeds were ground before freezing using a blender (BOSCH MKM6000, 180 W, Ljubljana, Slovenia).

2.2. Raman Instrumentation

Raman microspectroscopy was focused on the direct analysis of seeds which were longitudinally cut at room temperature prior to analysis. Spectra were recorded in the range 200–1800 cm⁻¹, using an XploRA Raman spectrometer (Horiba Jobin Yvon, Kyoto, Japan) following the procedure described in the literature [12]. The spectra preprocessing was realized using Spectragryph software, version 1.2.13 [13].

2.3. Extraction of Oil Fraction and Fatty Acid Analysis

Prior to fatty acid (FA) analysis, the ground seeds were defrosted. About ~2.5 g was weighed on an analytical balance and transferred into glass vials, and then 7 mL of n-heptane was added in order to extract FAs. The extraction of fatty acids from the rosehip seeds was performed at room temperature (~23 °C) using ultrasound-assisted extraction (UAE) for 1.5 h on an ultrasound instrument (Vabsonic SB-8L T, Niš, Serbia) with a volume of 1 L, a frequency of 40 kHz and an input power of 30 W. After the extraction was complete, the sample was filtered through quantitative filter paper (pore size 2–4 μm), and the solvent was evaporated. The mass of the oil fraction was measured after the solvent removal and expressed in percent (%).

After solvent removal, the oil fraction was derivatized with BF₃/MeOH reagent (Supelco, Darmstadt, Germany) to convert FAs into fatty acid methyl esters (FAMEs), which were analyzed by gas chromatography with a flame ionization detector (GC-FID) using an Agilent Technologies 6890 (Santa Clara, CA, USA) instrument as described in the literature [14]. The content of FAs was identified by comparing the retention times with the peaks of the analytical standard acid mix containing 37 FAMEs(Supelco, Steinheim, Germany).

3. Results and Discussion

3.1. Raman Spectroscopy Analysis

With Raman spectroscopy, we demonstrate the fast and non-destructive characteristics of the method on seeds of rosehip. The chemical composition of Rosa sp. seeds was evaluated based on bands recorded in the region 200–1800 cm⁻¹ (Figure 2).

The fingerprint region of Raman spectra in Rosa canina seeds includes essential bands which correlate with the most important parts of the fatty acids’ molecular structure [15]; the region is also well known to characterize the unsaturation level of the fatty acid chain [16]. The predominant fatty acids detected in the rosehip seeds are unsaturated fatty acids (UFAs) (linolenic, linoleic and oleic), with the highest percentage of linoleic acid [2] and a lower percentage of saturated fatty acids (SFAs) (palmitic and stearic) [9]. In the Raman spectra of linolenic, linoleic and oleic acid, there are three or two broad C=C bonds with higher wavenumbers [17]. These acids mainly differ in the position of the double bond; consequently, their Raman spectra are highly similar [18]. The bands at 1655 and 1266 cm⁻¹, observed in the seed spectrum (Figure 2), are related to the presence of unsaturated fats and can be assigned to the cis stretching vibration of C=C and the bending of C-H, respectively [11]. All involve the unsaturation moieties of unsaturated fatty acid cis isomers, whose relative intensity is in accordance with the degree of saturation of the fatty acids in the lipid, especially in the case of the band at 1655 cm⁻¹ [17]. The lowest-intensity band at 1729 cm⁻¹ (Figure 2) was assigned to the stretching of C=O from the triacylglycerol structure that was present in all Raman spectra of different plant oil samples [17].

The presence of phenolic compounds in Rosa seeds was indicated by higher-intensity signals at 1598 and 369 cm⁻¹ (Figure 2), which primarily originate from the lignin of seed cell wall compounds [19,20]. Additionally, the higher intensity bands such as 1091 and 1120 cm⁻¹ in the spectrum (Figure 2) could be assigned to polysaccharides due to C-O-C, and 1458 cm⁻¹ due to C-O-H stretching vibrations of carbohydrates [10,11].

3.2. Fatty Acid Content

Lipids are considered one of the most fundamental constituents in human nutrition. Fatty acids are major constituents of lipids, and essential fatty acids (EFAs) such as ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) have to be acquired from the diet. Additionally, these FAs have been considered as functional foods and nutraceuticals. Many studies have delineated their significant roles in many biochemical processes, resulting in health promotion activities [21,22]. Most naturally occurring UFAs have the cis configuration, while FAs with the trans configuration occur in products as a result of technology processing (i.e., hydrogenation) [21].

The content of oil in rosehip seeds is low, up to 15%. The extraction procedure affects the oil yield, and the modern methods usually provide higher yields compared to Soxhlet extraction [5,22]. In this study, the yield of the seed lipid fraction obtained by the application of ultrasound-assisted extraction (UAE) was 5.6% (0.14 g per 2.5 g of seeds), which is in line with literature data for other extraction methods [5,22]. The modern extraction methods such as ultrasound, microwave and subcritical fluid extraction are utilized for obtaining higher-quality oils [5].

The fatty acid content was calculated as mg/g lipid and expressed as a relative amount in percent (%) of total FAs. The results reveal that the most abundant FA in the studied rosehip seed oil sample was arachidic acid (32.93%), followed by linoleic acid (29.72%), heneicosanoic acid (19.27%), palmitoleic acid (7.02%), α-linolenic acid (4.20%), oleic acid (4.01%) and behenic acid (2.85%) (Figure 3).

Among the detected FAs, two acids belong to omega-7 (palmitoleic acid) and omega-9 (oleic acid) monounsaturated fatty acids (MUFAs), whereas linoleic acid and α-linolenic acid are ω-6 and ω-3 PUFAs, respectively. The relative content of SFAs was somewhat higher compared to UFAs (Figure 3B). The unusually high percentage of arachidic acid might be a consequence of storage conditions as well as the applied extraction method.

The results obtained in this study for UFAs are in line with the literature, where it is reported that the most abundant ones are linoleic, oleic, linolenic and α-linolenic acid in seeds of rosehip (R. canina L.) originating from different regions of the world. On the other hand, variability in the qualitative and quantitative composition of FAs in seeds is well documented [2,4,6,7,23,24,25]. Data about the chemical composition and FA profiles of rosehip seeds could indicate that differences may result from the influence of numerous factors such as climatic, environmental and genetic factors.

4. Conclusions

This study confirms the successful application of Raman spectroscopy in the detection of lipids and fatty acids in seed storage reserves of rosehips in a straightforward and fast manner. The bands in the spectrum clearly indicated the presence of cis UFAs, and GC analysis confirmed that linoleic acid was the most abundant one. Raman spectroscopic analysis also detected phenolic compounds and polysaccharides in seeds.