Evaluation of Flavonoids and Furanocoumarins from Citrus bergamia (Bergamot) Juice and Identification of New Compounds

Bergamot juice (BJ) contains different classes of flavonoids (e.g. flavanones and flavones) that can exert beneficial effects on human health. The aim of this study was to evaluate the qualitative and quantitative composition of a BJ obtained from fruits harvested in Southern Italy (Calabria) at the end of their maturation period. The identity of several flavonoids and furanocoumarins was assessed by co-chromatography, UV spectra and molecular weight comparison. The unknown compounds were dissociated by induced collision (CID-MS) and their identity established through the characteristic ions product. By this approach a complete profile of about twenty compounds (furano-coumarins, flavonoids C- and O-glycosides) present in BJ was obtained. Furthermore, three acylated flavanones, present in amounts of 20.1±1.1, 89.3±2.2 and 190.1±3.1 mg/L, respectively, and which seem to correspond to di-oxalate derivatives of neoeriocitrin, naringin and neohesperidin, were identified for the first time in BJ. The other main flavanones were naringin, neohesperidin and neoeriocitrin, and their content was 167.5±1.8, 123.9±1.7 and 73.3±1.6 mg/L, respectively. Concerning flavones, the total amount in BJ was about 160 mg/L and the main ones were vicenin-2, stellarin-2, rhoifolin and neodiosmin. Bergapten and bergamottin were the primary furanocoumarins in BJ and their amounts were 9.0±0.4 and 18.2±0.5 mg/L, respectively.


Introduction
Flavanones represent a small group of compounds present in high concentrations in different citrus like orange, grapefruit and lemon and in lesser amounts also in tomatoes, some aromatic plants and propolis. In citrus juice the main aglycones are naringenin, hesperetin and eriodictyol glycosylated with neohesperidose, which imparts a bitter taste, or rutinose that is flavourless. Bergamot (Citrus bergamia Risso) fruit and juice (BJ) are a source of bitter taste compounds containing mainly the flavanones neohesperidin, naringin, neoeriocitrin and lesser amounts of flavones and furanocoumarins. After ingestion as food the flavanone glycosides are metabolised by human intestinal bacterial microflora to the respective aglycones [1,2], which seem to possess antioxidant [3], anticarcinogenic [4], hepatolipidemic [5] and anti-inflammatory [6] activities. In addition, naringenin and hesperetin can bind to estrogen receptors [7], or inhibit the activity of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens [8]. Naringin was found to lower total cholesterol and lowdensity lipoprotein cholesterol levels in plasma [9], and hesperidin to also significantly lower plasma triglyceride levels [10]. Chronic administration of BJ caused in rats a significant reduction in serum cholesterol, triglycerides, LDL and an increase in HDL levels [11]. Moreover, the authors observed a protective effect on the hepatic parenchyma. Bergamot juice contains diverse compounds with similar chemical structure thus a sophisticated high resolution technique is necessary for their identification and quantification. For this reason liquid chromatography coupled to DAD and mass spectrometry is the best choice for the analytical characterization of citrus [12][13][14][15]. The aim of this study was (1) to develop a LC-DAD-ESI-MS(MS) method allowing the qualitative and quantitative evaluation of flavonoids and furanocoumarins and (2) to identify unknown polyphenol compounds in the bergamot juice by LC-MS 2 .

Results and Discussion
The analyzed BJ contained different classes of compounds like flavonoids, cinnamic acid derivatives and furanocoumarins, whose structures are given in Figure 1. Bergamot juice is a complex food matrix and the identity of its components can be correctly assessed by LC coupled with DAD and a tandem mass spectrometer. A good separation of flavonoids in BJ was achieved with a Luna(2) C 18 narrow bore column with gradient elution. Figure 2 Table 1. The amount of flavonoids and furanocoumarins were determined using calibration curves obtained with standards, while for compounds X1, X2-X3 caffeic and ferulic acid calibration curves were used, respectively. Their amounts were then normalized by the molecular mass ratios. The amount of flavonoids determined in this paper agrees with those reported in literature [17], but they are not in agreement with other studies indicating higher amounts of flavanones [14]. The discrepancy could be ascribed to different factors such as: BJ preparation procedure, BJ obtained from various cultivars, different maturation period or the use of unspecific chromatographic methods not able to separate neoeriocitrin, naringin and hesperidin from their di-oxalate derivatives. Regarding furanocoumarins, we found in BJ bergamottin amounts lower than that reported in literature [17].  Regarding the validation method, calibration curves were constructed for each standard at five concentration levels and three independent determinations were performed at each concentration. The recovery of the extraction from spiked BJ samples was 97.2±3.1. The precision of the method was tested by both intra-day (n=5) and inter-day (5 days, n=5) reproducibility, and the coefficient of variation was below 3.3 %. Limit of quantization and limit of detection were 2 μg/ml and 1 μg/ml, respectively.

Conclusions
Bergamot juice contains several different compounds and LC-DAD-MS is the best choice for their separation, identification and quantization. Moreover, tandem mass spectrometry with collision induced dissociation allows structural identification, especially when standard compounds are not available.

Method validation
The LC-DAD-MS method was validated for linearity, LOQ, LOD, accuracy, peak purity, precision and repeatability. Lower limit of quantization (LLOQ, S/N ratio of 8) and lower limit of detection (LLOD, S/N ratio of 3) were determined by serial dilution of standard solutions. Accuracy was evaluated by spiking three BJ sample with four amounts (2, 5, 10 and 20 μg/mL) of the mix standard compounds. Peak purity and identity were confirmed by LC-DAD-MS and LC-MS 2 experiments. Precision (intra-and inter-day) of the assay was verified by analyzing BJ samples 3 times on five consecutive days. Repeatability was confirmed by evaluating standard deviations of the retention times and peaks area.

Sample preparation
Bergamot fruits (1 Kg) were peeled, squeezed and the obtained juice was mixed and apportioned in 20 mL containers, which were stored at -80°C. Ten mL were diluted 5-fold in methanol-formic acid 1% (90:10, v/v) and the resulting solution was sonicated for 10 min and then centrifuged at 1000 x g for 10 min. One aliquot of the supernatant was filtered through a Millipore 0.2 μm disk and 5 μL were injected into the LC system.

LC-DAD-MS/MS analysis
The chromatographic system was an Alliance mod. 2695 (Waters, Milford, MA) equipped with a mod. 2996 (Waters) photodiode array detector and a triple quadrupole mass spectrometer mod. Quattro micro (Micromass, Beverly, MA). A 3 μm C 18 Luna (2) narrow bore column (150 x 2.0 mm, Phenomenex, Torrance, USA) was used for the separation, which was performed by means of a linear gradient elution (eluent A, 0.1% formic acid; eluent B, acetonitrile) at a flow rate of 250 μL/min. The gradient was as follows: from 10 to 25% B in 20 min, 25% B for 5 min, 25 to 50% B in 5 min, 50 to 95% B in 5 min and then 95% B for 15 min. The column was maintained at 30°C. Chromatographic data were acquired in the 200-450 nm range and were integrated at 282 nm (flavanones) and 330 nm (flavones, furanocoumarins and cinnamic acid derivatives). The mass spectrometer operated in positive and negative full-scan mode in the range 100-1000 Da. The capillary voltage was set to 3.0 kV, the cone voltage was 20V, the source temperature was 130°C, and the desolvating temperature was 350°C. All data were acquired by Masslink 4.0 software (Micromass) with the Quan-Optimize option for the fragmentation study. Calibration curves were obtained from narirutin, naringin, hesperidin, neohesperidin, rhoifolin, neodiosmin, neoeriocitrin, bergapten, bergamottin, caffeic and ferulic acid stock solutions prepared by dissolving 5 mg of standard powder in 5 mL methanol. They were measured in the range of 2-20 μg/mL.