The scientific interest in plant phenolics as chemopreventive and therapeutic agents against chronic and degenerative diseases has been increasing since the late 1990s, when the French paradox was associated with the high intake of phenolics present in red wine [1
]. Ellagitannins and ellagic acid have been studied mainly for their positive effects on human health and for their physiological properties, such as anti-tumor properties [2
Research on the chemical characterization of phenolic compounds in R. rugosa
, their stability in food processing techniques and storage was an area of our interest. In Poland, Rosa rugosa
was introduced in 1960 [3
]. Every part of the Rosa rugosa
plant contains large amounts of phenolic constituents [4
]. The petals are rich in hydrolysable tannins, from which their medicinal properties are believed to derive. Two ellagitannins present in wrinkled rose petals, tellimagrandin I and pedunculagin, have been demonstrated to inhibit human immunodeficiency virus type-1 reverse transcriptase (HIV-1RT) in vitro [8
]. Polyphenols isolated from R. rugosa
petals have also been shown to have antioxidant properties [4
]. Liu L. et al [14
] suggests that polyphenol-enriched extract of R. rugosa
reduced blood glucose in type 2 diabetic rats by an improvement of insulin sensitivity. Certain phenolic compounds from wrinkled rose petals inhibited lipid peroxidation [15
]. It has been found that the polyphenols present in beverages of low alcohol content can effectively neutralize the radicals formed during its metabolism in the body [17
Liqueurs constitute an important group of spirit beverages on the global market, which represent a wide range of traditional drinks, among them Polish ‘nalewka’, a traditional Polish category of alcoholic beverage similar to medicinal tinctures. According to the Regulation of the European Union, liqueurs are spirit drinks produced by flavoring ethyl alcohol or a distillate of agricultural origin from foodstuffs such as fruit, herbs, wine, or other agricultural products, and sweetened, with minimum 15% v/v content of alcohol (Regulation [EC] No. 110/2008). The variety of liqueurs depends primarily on the geographical origin and climatic conditions of the region in which they are produced, which affects the choice of ingredients for the production of liqueurs.
Many innovative green extraction techniques have been developed to improve the phenolic compounds from plant material, such as pressurized liquid extraction, supercritical fluid extraction, microwave- or ultrasound or enzyme- assisted extraction [19
], hydrotermal extraction [24
], and far-infrared radiation [25
]. They could increase the extraction yield and decrease the extraction time, but expensive equipment seriously limits their application. For this reason we decided to choose a conventional method of liqueur manufacturing—maceration—utilized for more than a century for the isolation of polyphenols. Using aqueous ethanol, a common bio-solvent, easily available in high purity and completely biodegradable during maceration, we utilized elements of green extraction of a natural product.
In the available literature, there is little research on the phenolic compounds in rose liqueurs [26
]. Ellagitannins represent a less studied group mainly due to their diversity and chemical complexity. The first methodologies used for ellagitannin quantification were based on the capacity of tannins to form complexes with proteins, however, these methods are very unspecific. Spectrophotometric assays were developed generally using as the key reactants potassium iodate, pyridine, sodium nitrate, and methanolysis [27
]. Chromatographic methods used to quantify ellagitannins and ellagic acid include high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) (not considered as a quantification method) and electrophoresis chromatography, which shows low reproducibility [29
]. HPLC, a precise and robust separation technique, is the most adequate for the separation and quantification of ellagitannins. Most techniques have focused on the run conditions of HPLC with deficiencies in the extraction conditions and on sample pre-treatment [31
]. As the current concern is to obtain clean techniques and to use less solvent to meet the demands of green chemistry, UPLC is used for polyphenol analysis, allowing higher efficiency and resolution and shorter run times. The best way to define the qualitative composition of ellagitannins is using mass spectrophotometry in tandem with UPLC or nuclear magnetic resonance [33
According to Borràs et al. [38
], new analytical techniques are widely used for authenticity tests of spirit beverages, quality control, and adulteration assessments of foodstuffs. For this reason, the UPLC-PDA-Q/TOF-MS method was applied in the analysis of polyphenolic compounds in terms of the profiling of the polyphenols in rose liqueurs. Ellagitannins represent a less studied group mainly due to their diversity and chemical complexity. The analysis of phenolic compounds presents some challenges because of the enormous structural diversity of phenols, the number of possible isomeric structures, the complexity of sample matrices, the limited number of commercially available standards, and the lack of comprehensive high-performance liquid-chromatography–mass-spectrometry libraries for confident compound identity confirmation [39
The aim of the study was the identification and quantitative analysis of the polyphenolic compounds in liqueurs made from R. rugosa petals, collected from the “Polska Róża” plantation located in Kotlina Kłodzka, Poland. Studies were carried out on liqueurs after preparation and after 120 days of seasoning at room temperature without light. The identification and quantitative determination of the polyphenolic compounds in tests was carried out using the UPLC-PDA-Q/TOF-MS method.
Formic acid (98–100%) and acetonitrile (gradient grade for HPLC) were purchased from Sigma-Aldrich (Steinheim, Germany). (+)-Catechin, ellagic acid, isorhamnetin 3-O-glucoside, kaempferol 3-O-galactoside, myricetin 3-O-glucoside, quercetin 3-O-glucoside and sanguine H-2 and standards were purchased from Extrasynthese (Lyon, France). All reagents were analytical grade.
3.2. Plant Material
Fresh petals of Rosa rugosa was collected from the plantation of the company “Polska Róża” located in Kotlina Kłodzka (16°39′ E 50°27′ N, Poland) in June 2012. The collected petals were completely stained, gathered ripe, without signs of deterioration and mechanical damage. Fresh raw material was stored under refrigeration (6 ± 2 °C), until the time of analytical determinations, but no longer than three days.
3.3. Liqueurs Preparation
The research material constituted 12 variants of alcoholic liqueurs from petals of Rosa rugosa
differing by the concentration of ethyl alcohol applied for extraction, the acidity, and the addition of saccharose. The process of preparing the liqueurs consisted of a stage of preparing the extract and then the finished product. 50.00 g of rose petals (Rosa rugosa)
was weighed and covered with 500 cm3
ethyl alcohol of varying potency 40% or 65% (v
), depending on the process variation, without or with the addition of citric acid (in an amount 0.8 g/100 cm3
) to adjust the acidity. Maceration was conducted in sealed jars, without light, at room temperature (22 ± 2 °C) for 18 days. The contents of the jars were stirred every day. After 18 days, the extracts were vacuum filtered, and the volume and weight were measured. The liqueurs were prepared using the appropriate proportion by weight of extract, sucrose, water and citric acid in relation to the weight of the extract. Alcoholic, unsweetened and sweetened liqueurs were marked by the following codes for identification: A40_1, A65_1 (three parts of the extract + two parts of water); A40kw_1, A65kw_1 (three parts of the acidified extract + two parts of water); A40_1K, A65_1K (three parts of the extract + 1.96 part of water + 0.04 part of citric acid); A40_1C, A65_1C (three parts of the extract: one part of sucrose + one part of water); A40kw_1C, A65kw_1C (three parts of the acidified extract + one part of sucrose + one part of water); A40_1C+K, A65_1C+K (three parts of the extract + one part of sucrose + 0.96 part of water + 0.04 part of citric acid). A diagram of the technological operations is shown in Figure 1
and Figure 2
. The experiment was carried out in two technological duplicates. Studies were carried out on the liqueurs after preparation and after 120 days of seasoning at room temperature without light.
3.4. Qualitative and Quantitative Analysis of Polyphenolic Compounds by UPLC-PDA-ESI-QTOF-MS
The identification and quantification of the polyphenols was carried out with the use of an ACQUITY Ultra Performance LC system equipped with photodiode array detector with a binary solvent manager (Waters Corporation, Milford, MA, USA) series with a mass detector G2 Q/TOF micro mass spectrometer (Waters, Manchester, U.K.) equipped with an electrospray ionization (ESI) source operating in negative and positive modes [54
]. Separations of individual polyphenols were carried out using a UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 30 °C. The samples (10 µL) were injected, and the elution was completed in 15 min with a sequence of linear gradients and isocratic flow rates of 0.45 mL/min. The mobile phase consisted of solvent A (2.0% formic acid, v
) and solvent B (100% of acetonitrile).
The program began with isocratic elution with 99% solvent A (0–1 min), and then a linear gradient was used until 12 min, lowering solvent A to 0%; from 12.5 to 13.5 min, the gradient returned to the initial composition (99% A), and then it was held constant to re-equilibrate the column. The analysis was carried out using full-scan, data-dependent MS scanning from m/z 100 to 1500. Leucine enkephalin was used as the reference compound at a concentration of 500 pg/µL, at a flow rate of 2 µL/min, and the [M − H]− ion at 554.2615 Da was detected. The [M − H]− ion was detected during 15 min analysis performed within ESI–MS accurate mass experiments, which were permanently introduced via the LockSpray channel using a Hamilton pump. The lock mass correction was ±1.000 for the mass window. The mass spectrometer was operated in negative ion mode, set to the base peak intensity (BPI) chromatograms, and scaled to 12,400 counts per second (cps) (100%). The optimized MS conditions were as follows: capillary voltage of 2500 V, cone voltage of 30 V, source temperature of 100 °C, desolvation temperature of 300 °C, and desolvation gas (nitrogen) flow rate of 300 L/h. Collision-induced fragmentation experiments were performed using argon as collision gas, with voltage ramping cycles from 0.3 to 2 V.
The data obtained from UPLC–MS were subsequently entered into the Mass-Lynx™ 4.0 ChromaLynx Application Manager software. The runs were monitored at the following wavelength: ellagitannins at 254 nm, flavan-3-ols at 280 nm, phenolic acids at 320 nm and flavonol glycosides at 360 nm. The PDA spectra were measured over the wavelength range of 200–800 nm in steps of 2 nm. The retention times and spectra were compared to used standards. The quantification of phenolic compounds was performed by external calibration curves, using reference compounds selected based on the principle of structure-related target analyte/standard (chemical structure or functional group). The calibration curve of myricetin 3-O
-glucoside was used to quantify myricetin 3,5-di-O
-glucoside. The calibration curve of quercetin 3-O
-glucoside was used to quantify quercetin 3,4-di-O
-glucoside, quercetin 3-O
-glucosyl-xyloside, quercetin 3-O
-rhamnoside and unknown quercetin derivatives. The calibration curve of kaempferol 3-O
-galactoside was used to quantify kaempferol 3,4-di-O
-glucoside and kaempferol 3,7-di-O
-rhamnoside. The calibration curve of isorhamnetin 3-O
-glucoside was used to quantify isorhamnetin 3-O
-glucoside. (+)-Catechin was quantified with the (+)-catechin standard. The calibration curve of sanquine H-2 was used to quantify sanquine H-2, unknown ellagitannins and isomer galloilo-bis-HHDP glucose. The calibration curve of ellagic acid was used to quantify ellagic acid (Table 4
). All determinations were done in triplicate (n
3.5. Method Validation
The method was validated in accordance with the requirements for new methods for linearity, limit of detection (LOD), limit of quantification (LOQ), precision (interday and intraday precision), repeatability and stability (Table 4
Standard calibration curves were prepared using the following standards: (+)-catechin, ellagic acid, isorhamnetin 3-O-glucoside, kaempferol 3-O-galactoside, myricetin 3-O-glucoside, quercetin 3-O-glucoside and sanguine H-2. Standard stock solutions were diluted to appropriate concentrations (five calibration points were used in each case) for the plotting of calibration curves. The linearity was obtained by plotting the peak areas versus the corresponding concentrations (µg/mL) of each analyte.
3.5.2. LODs and LOQs
The LOD and LOQ of standard stock solutions were determined by preparing dilute solutions of standards (five dilution points were used in each case), and injecting these solutions into the liquid chromatograph and recording the signal-to-noise (S/N) ratio for peaks at each concentration. LODs and LOQs were determined at an S/N ratio of about three and 10, respectively.
3.5.3. Precision, Repeatability and Stability
For the intraday precision test, the standard solution containing the seven standard compounds were analyzed three times within one day (n = 3), while for the interday precision test, the standard solution was examined each day for three consecutive days (n = 9). The intraday and interday precision of the current method was evaluated by calculating the relative standard deviation (RSD, %) of the peak areas.
For repeatability, three different sample solutions (n = 3) prepared from the same sample were analyzed, and variations were expressed by RSD (%).
For stability investigation, sample solutions were analyzed each day for three consecutive days (n = 9) and variations were expressed by RSD (%).
3.6. Statistical Analysis
All results are expressed as the mean ± standard deviation (SD) of three replicates. Statistical analysis was conducted using Statistica version 12.0 (StatSoft, Tulsa, OK, USA). Significant differences (p < 0.05) between average responses were evaluated with the use of one-way analysis of variance (ANOVA) with Duncan test.
A green analytical UPLC-PDA-Q/TOF-MS method can be applied as a rapid, reliable and sensitive tool for the quality control of liqueurs, and the identification and quantification of the major polyphenols. Applied UPLC enabled a reduction of time analysis and solvent consumption.
We demonstrated that a high stability of polyphenols in rose petals in alcoholic liqueurs in various technological variants during seasoning is consistent with the literature data. It is confirmed that the extraction of polyphenols from the petals using acidified aqueous ethanol as a solvent is more effective than aqueous ethanol at the same concentration. The obtained results show that the profile and content of the polyphenolic compounds, which are a feature of their health-promoting properties, do not change during liqueur seasoning. Maceration still remain the most common extraction technique for natural products.
The experimental results confirmed the hypothesis that R. rugosa petals are rich in phenolic compounds, particularly in ellagitannins 1517.01 mg/100 g FW.
Rosa rugosa petals can be a valuable raw material for food industries as a source of not only colours and flavors, but also bioactive compounds for dietary supplements or functional foods.