Volatile and Flavonoid Composition of the Peel of Citrus medica L . var . Corsican Fruit for Quality Assessment of Its Liqueur

Citrus fruits have an important role in the world economy, and in local regions of the Mediterranean area, such as Corsica, they are consumed either directly or processed. The citron (Citrus medica L.) belongs to the family Rutaceae, subfamily Aurantiaca, tribe Citrae (1). Systematic studies highlight the major role of citron in the phylogeny of several varieties of the Citrus genus as the pollinator parent (2–6). All taxonomists recognize the contribution of the citron to the development of several important cultivated genotypes. For instance, citron combined with sour orange (C. aurantium) and C. micrantha is phylogenetically the ancestor of lemon (C. limon) and lime (C. aurantifolia), respectively (7,8). Citron trees are small, 3 to 5 m high, with thorny branches and oval, elongated leathery leaves. Considered indigenous to northeastern India, Myanmar, and Yunnan province of China, the citron was the fi rst Citrus species introduced to the Mediterranean basin.


Introduction
Citrus fruits have an important role in the world economy, and in local regions of the Mediterranean area, such as Corsica, they are consumed either directly or processed.The citron (Citrus medica L.) belongs to the family Rutaceae, subfamily Aurantiaca, tribe Citrae (1).Systematic studies highlight the major role of citron in the phylogeny of several varieties of the Citrus genus as the pollinator parent (2)(3)(4)(5)(6).All taxonomists recognize the contribution of the citron to the development of several important cultivated genotypes.For instance, citron combined with sour orange (C.aurantium) and C. micrantha is phylogenetically the ancestor of lemon (C.limon) and lime (C.aurantifolia), respectively (7,8).Citron trees are small, 3 to 5 m high, with thorny branches and oval, elongated leathery leaves.Considered indigenous to northeastern India, Myanmar, and Yunnan province of China, the citron was the fi rst Citrus species introduced to the Mediterranean basin.
During the 1890s, Corsica was the world's leading citron producer, exporting 1700 tonnes of fruit per year.The brined fruits were shipped mainly to northern Europe and used as candied peel in traditional Christmas cakes.In Corsica, only 5 or 6 ha of citron were still cultivated in 2008; the variety grown is Corsican.The fl owers of this variety are white, while those of other citron varieties are usually purple or pink.The Corsican variety is the only one of the citron group that has acid-free pulp (juice vesicles) and sweet albedo.The fruit of the Corsican variety is generally light orange-yellow to cadmium yellow, large and elliptical in shape, 7-10 cm i.d., 8-14 cm in length, with a rind about 3-4 cm thick.The citron's highly scented peel is a ributed to its external vesicles, which contain es-sential oils, rather than its pulp, which is seldom consumed.Its skin is thick, and its white inner tissue, called albedo, is consumed usually a er being processed into jam or candied fruit.In Corsica, the main product from the peel is a local liqueur (called Cédratine in French or Aliméa in Corsican language).For the production of this alcoholic beverage, the harvest of citron fruits runs usually from September to November.A second set of fruits can be also collected from January to February.
Several chemical studies have reported the volatile composition of peel from C. medica.The peel essential oil composition from various citron varieties has also been investigated: Etrog, Diamante, Rhobs el Arsa, Buddha's hand and Corsican (9)(10)(11)(12)(13)(14).These studies showed quantitative diff erences with regard to the major constituents: limonene, γ-terpinene, geranial and neral.Thus, several chemical compositions of the same variety have been reported according to their geographical origins.Venturini et al. (15) reported the chemical compositions of peel and leaf oils from 17 citron cultivars.According to the levels of seven components (limonene, β-pinene, γ-terpinene, neral, geranial, nerol and geraniol), the citron cultivars were classifi ed in four main oil chemotypes.To our knowledge, there have been no studies on the polyphenolic composition of C. medica var.Corsican.However, some articles have dealt with fl avonoid components in other varieties of C. medica (16)(17)(18).The fl avonoids reported in the fruits are rutin and hesperidin derivatives (sakuranetin, 7-O-methyl-aromadendrin, dihydrokaempferide).High amounts of glycosylated fl avones, fl avanones, and dihydrochalcone were also identifi ed; the major compounds were C-and/or O-glycosides of apigenin, phloretin, diosmetin and hesperetin.
As part of our investigations on the chemical characterization of citron varieties (15) and alcoholic beverages from Corsica (19), the aim of this work is to provide a better knowledge of the chemical characteristics (volatile and polyphenolic compositions) of typical commercial liqueurs from C. medica var.Corsican.This characterization should lead to a be er valorization of these products by standardization of their quality.

Sampling of plant material
One thousand and fi ve hundred fruits of Citrus medica var.Corsican (bourgeoning stage) were selected from 100 trees grown under the same pedoclimatic conditions.All trees were grown on a plantation at Vescovato, Corsica, France (latitude 42° 29' N, longitude 9° 30' E, Mediterranean climate, soil derived from alluvial deposits and classifi ed as fersiallitic).Citron fruits were collected each month from July to January.Phenotypic characteristics of fruits such as mass, length, width, and colour were measured each month.These fruit samples were also used for the preparation of peel essential oil and solvent extracts at each harvest.

Determination of fruit maturity
The degree of maturity of Corsican citron was evaluated using the method described by Stenzel et al. (20) and based on the calculation of growing degree-days (GDD).It should be noted that this procedure provides results independent of interannual variations of temperature, but dependent on the species or varieties and environmental factors.The fl owers of C. medica var.Corsican were scored at the date of anthesis (fl owers blooming on April 15, 2010).From this date until the fi nal harvest of the fruit (January 4, 2011), the air temperature was measured daily by recorders placed on the canopy of three trees (facing east).

Isolation of essential oil
The peel of C. medica L. var.Corsican was water distilled (5 h) using a Clevenger-type apparatus (Midisciences, Fuveau, France) according to the method recommended in the European Pharmacopoeia and the essential oil yields were 0.40-0.72 %.Each distillation was performed in triplicate.

Preparation of solvent extracts
A mass of 100 grams of fresh peel was lyophilised and extracted at room temperature with ethanol (1 L).The solution was fi ltered on a Büchner funnel and extracted with ethyl acetate (3×100 mL).The solution was evaporated, recovered in methanol (50 mL) and fi ltered through a 0.45--µm membrane (Phenomenex, Le Pecq, France).Each polyphenol extraction was performed in triplicate.

Essential oil analysis
Gas chromatographic analysis was carried out using a Perkin-Elmer (Waltham, MA, USA) Autosystem XL apparatus equipped with dual fl ame ionization detection (FID) systems and two fused silica capillary columns (60 m×0.22 mm i.d.; fi lm thickness 0.25 µm) coated with Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol).The oven temperature was programmed from 60 to 230 °C at 2 °C/min and then held isothermally at 230 °C for 35 min.The injector and detector temperatures were maintained at 280 °C.Samples were injected in the split mode (1/50) using helium as carrier gas (1 mL/min); injection volume of pure oil was 0.2 µL.For headspace sampling by solid--phase microextraction and gas chromatography (HS--SPME-GC) analysis, only the Rtx-1 column was used and volatile components were desorbed in a GC injector with an SPME Intel liner (0.75 mm i.d.; Supelco, Bellefonte, PA, USA).Samples were also analyzed with a Perkin-Elmer Turbo mass detector (quadrupole), coupled to a Perkin--Elmer Autosystem XL, equipped with fused silica capillary columns Rtx-1 and Rtx-Wax.Ion source temperature was 150 °C, energy ionization was 70 eV, electron impact mass spectra were acquired over the mass range of 35-350 Da (scan time: 1 s).Other GC conditions were the same as described above except for the use of a split ratio of 1/80.For HS-SPME-GC-mass spectrometry (MS) analysis, only the Rtx-1 column was used and volatile components were desorbed in a GC injector with an SPME Intel liner (0.75 mm i.d.; Supelco).The identifi cation of individual volatiles was based on comparison of calculated retention indices (apolar and polar columns) and mass spectra with those of our own library of authentic compounds or literature data (I l ) (21).Retention indices (I a and I p , respectively) of the compounds were determined relative to the reten-tion times of series of n-alkanes (C 5 -C 30 ) with linear interpolation, using the Van den Dool and Kratz equation (22) and so ware from Perkin-Elmer.The relative concentrations of components were calculated based on the GC peak areas (apolar column Rtx-1) without using correction factors.

Analysis of solvent extract
Solvents and reagents used for sample preparation and chromatography were LC-MS grade acetonitrile (ACN) and ammonium acetate (NH 4 OAc, LC-MS grade) obtained from Fisher Scientifi c (Illkirch, France).Deionized water was purifi ed using a MilliQ water (Millipore, Bedford, MA, USA) purifi cation system.Reference fl avonoids (99 % purity determined by high-performance liquid chromatography (HPLC) were purchased from Extrasynthese (Geney, France).Solutions of reference compounds were prepared by dissolving the compounds in ACN at 1 mg/mL and then fi ltered through a 0.2-µm polytetrafl uoroethylene (PTFE) fi lter.Direct-infusion MS analyses were performed with reference solutions at a concentration of 0.1 mg/mL in volume ratio of ACN/ H 2 O=5:5 and 0.1 % NH 4 OH.Prior to LC-MS/MS analysis, the mixture was diluted in the initial mobile phase to obtain the desired concentrations.
The LC system consisted of a Flexar ultra-high-performance liquid chromatography (UHPLC; Perkin-Elmer) with two Flexar FX-10 UHPLC pumps, a Flexar solvent manager, a 275-Flexar autosampler, and a Flexar LC PE200 column oven.UHPLC analyses were performed on a 100 mm×2.1 mm i.d., 3 µm, LUNA 3U C18 column (Phenomenex) and the column temperature was set at 30 °C.A volume of 10 µL of sample was injected using an injection loop of 15 µL in full loop mode.The mobile phase consisted of MilliQ water (solvent A) and ACN (solvent B) with 0.1 % (by volume) NH 4 AcO acetate buff er at a fl ow rate of 500 µL/min.The column was equilibrated in 90 % A and 10 % B for 5 min, and elution was carried out with the following linear gradient from 90 to 10 % A in 2 min, an increase from 10 % A to 100 % in 14 min, and 100 % A for 6 min.
Mass spectra were acquired using an AB Sciex (Toronto, Canada) 3200 QTRAP linear triple quadrupole fi tted with an electrospray ionization (ESI) source operating in positive or negative mode.High purity nitrogen was used as both nebulizer and turbo gas.The ESI source was operated with following se ings in positive mode: curtain gas (CUR) 25 psi, nebulizer gas (GS1) 31 psi, heater gas (GS2) 65 psi, ion spray voltage (IS) 5000 V and temperature 550 °C.The following se ings were used in negative mode: CUR 25 psi, GS1 41 psi, GS2 65 psi, IS -4200 V and temperature 550 °C.For enhanced product ion (EPI) experiments, the MS parameters were set as follows in positive ion mode: declustering potential (DP) 50 V, entrance potential (EP) 10 V, collision energy (CE) -35 V, and collision energy spread (CES) ±15 V, and in negative mode: DP -80 V, EP -10 V, CE -35 V, and CES ±15 V.The so ware used for data acquisition and data analysis was Analyst v. 1.5.1 (AB Sciex).For each reference compound, a relevant transition of the pseudomolecular ion was selected and the mass parameters were optimized in direct infusion (fl ow rate: 10 µL/min) using the automated component optimization function of the Analyst so ware.Multiple EPI spectra were recorded in our MS spectral library for each compound.Data acquisition was performed in the multiple reaction monitoring (MRM) mode, followed by an EPI scan (MRM-EPI).EPI mass spectra were recorded in the range of m/z=50-1000 at 4000 Da/s.Compound identifi cation was allowed by comparison of retention time, MRM transition and the EPI mass spectrum of reference compounds.An external standard method was used for quantifi cation of fl avonoid compounds.The quantification was performed using a calibration curve obtained by serial diluted reference solution (six levels of concentrations with three injections per level) of each compound in our chromatographic conditions.

Identifi cation of fl avonoid components
The optimized MS/MS parameters (declustering potential, entrance potential, collision cell entrance potential, collission energy, collision cell, exit potential) and MRM transitions for each fl avonoid component of C. medica var.Corsican extract were described in Table 1.According to the data previously reported in literature (23)(24)(25)(26), the fragmentation mechanism of molecular ions to fragment ions used for MRM transitions was indicated in Table 1.

Analysis of commercial liqueur
Commercial citron liqueur prepared with C. medica var.Corsican peel (fruits harvested in November 2010) was provided by the Mavela distillery (Aleria, Corsica, France).These commercial liqueurs (10 mL of sample in a 20-mL vial) were subjected directly to HS-SPME.The SPME device (Supelco) coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) (fi bre 2 cm, 30 µm thick ness) was used for the extraction of volatile compounds from the liqueur.The extraction required the optimization of HS-SPME parameters (temperature and extraction times), based on the sum of total peak areas.The equilibrium time was 120 min.The temperature and time of extraction were selected a er three diff erent experiments at 20, 50 and 70 °C, respectively, for each diff erent extraction time (15, 30, 60 and 120 min).The maximum sum of total peak areas was obtained at 20 °C for 30 min for liqueurs.A er sampling, the SPME fi bre was inserted into the GC and GC-MS injection ports for desorption of volatile components (5 min at 250 °C), both using the splitless injection mode.Before sampling, each fi bre was reconditioned for 5 min in the GC injection port at 260 °C.HS-SPME and subsequent analyses were performed in triplicate.Volatile and fl avonoid analyses of commercial citron liqueurs were performed using the same experimental parameters as those described for essential oil and solvent extractions, respectively.

Fruit maturity according to growing degree-days
Table 2 shows the number of days per month for which the formulae A, B and C described by Stenzel et al. (20) were applied, the GDD for each month (April to De-cember), and the total GDD from the date of anthesis (April 15) to the harvest (July to January).The monthly GDD increased between April (68.2) and August (434.1),then decreased in the following months to reach 31.6 in December.The total GDD until the harvest date increased from June (705.0)until September (1927.7)and then took an asymptotic value (2105.5-2204.9) in the following months (October-December).Fruit maturity was obtained at a value of total GDD in the order of 2000.Stenzel et al. (20) showed that orange (C.aurantium) from Brazil requires 2500 to 3600 of total GDD to reach full maturity.This level of GDD does not seem to be necessary to obtain the maturity of the Corsican citron variety.This could be explained by the diff erence of climatic factors between Corsica (Mediterranean area) and Brazil (tropical area) or by the diff erence of metabolism between species (20).
The morphological characteristics (mass, length, and width) were measured at each harvest (Table 2), and they increased from July to October, a er which they remained stable from October to January.Immature fruits had a dark green colour (July-August), which changed to green-yellow (September-November) and to yellow (November--December) of mature fruits.A er maturity, citron fruits take a yellow-orange colour.Corsican citron fruit is traditionally collected in November.The mature fruit (November) has a mass of 1100-1300 g, length of 12-13 cm, and width of 12.5-13.5cm.

Analysis of volatile components
The essential oil composition of the peel of C. medica var.Corsican according to the development stage of fruit was studied monthly from July to January (Table 3).Thirty compounds were identifi ed in the peel essential oil (97.6-99.5 % of the total composition).Twelve monoterpene hydrocarbons, twelve oxygenated monoterpenes, four sesquiterpene hydrocarbons, and two linear oxygenated components were reported.Essential oil was dominated by monoterpene hydrocarbons (66.8-82.5 %).Limonene was the major compound (54.2-60.6 %) followed by During fruit development, the yield and the chromatographic profi le of essential oil exhibited signifi cant diff erences between immature fruits (July) and later harvested fruits (August to January).Fruit samples from July had an oil yield of 0.4 %, while the other samples exhibited yields near 0.7 %.The peel essential oil of C. medica var.Corsican showed a relatively stable chemical composition (Table 3) from August to January with a chromatographic profi le dominated by limonene (57.8-54.6 %) and γ-ter pinene (11.5-15.2%).The content of the major component, limonene, can be considered stable (54.2 % in July, 54.6 % in January).A signifi cant diff erence between immature and mature fruits was only observed in the content of γ-ter pinene.Relative percentages of γ-terpinene increased between July and the following months, whereas the content of nerol, neral, geraniol and geranial decreased in the same period.However, the decrease of nerol (5.9 % in July to 4.3 % in November) and neral (from 5.7 % in July to 4.7 % in January) is not related to fruit development (Table 3).
The HS-SPME volatile fraction of the commercial liqueur had seventeen components.All these compounds were also reported in Corsican citron essential oil.From a quantitative point of view, the volatile composition of citron peel oil and commercial liqueur showed several similarities, except for the essential oil from immature fruits (July).For instance, the major components were always limonene (60.5 %) and γ-terpinene (21.8 %).However, it was not possible to establish direct correlation between the chemical composition of the peel essential oil and that of the volatile fraction of liqueur obtained by HS-SPME.The two extraction processes (hydrodistillation vs. headspace) and HS-SPME parameters (particularly the temperature) could be responsible for the diff erence in the contents of oxygenated compounds.

Analysis of fl avonoid compounds
As far as we know, the fl avonoid composition of C. medica var.Corsican species has not been reported previously.Thirteen compounds were identifi ed including three glycosylated fl avanones, two glycosylated fl avonols, one glycosylated fl avone, one aglycone fl avone, three polymethoxylated fl avonols and three polymethoxylated fl avones (Table 4).The main chemical classes determined on dry mass basis were the glycosylated fl avonols (10.0-14.3mg/g) followed by glycosylated fl avanones (1.1-10.2mg/g) and fl avones (0.5-9.9 mg/g).
The main components were identifi ed as rutin, diosmin, and neohesperidin.Mass fractions of fl avonoids changed diff erently with fruit maturity.The rutin mass fraction was stable from July to January between 8.0 and 11.2 mg/g.The mass fraction of diosmin decreased gradually from July to January (9.9 to 0.5 mg/g, respectively).Conversely, the mass fraction of neohesperidin increased during fruit development from 0 (July) to 8.7 mg/g (January).The total concentration of fl avonoids decreased from July to September (total GDD <2000) changing from 26.5 to 13.8 mg/g and then increased in the following months (October-January, total GDD >2000), reaching values between 18.6 and 25.3 mg/g.From this result, it can be seen that the content of polyphenolic compounds varied during fruit development.Secondary metabolites may play a role in plant protection against photooxidative stress, in mediating thermotolerance and in direct defense against microbes and insects (30-32).
All polyphenolic compounds detected in the peel extract were also reported in the commercial liqueur.The major fl avonoid compounds of this product were rutin (16.8 mg/g) and neohesperidin (4.5 mg/g).Rutin and hesperidin were also reported as major compounds of solvent extract from C. medica var.Etrog (18).To the best of our knowledge, C. medica is the only Citrus species containing these as main components.Two main types of glycosylated fl avanones, neohesperidosides and rutinosides, had previously been reported in the peel compositions of other Citrus species (33).These two forms were identifi ed in C. medica extract with neohesperidin and rutin as major components.However, the fl avonoid composition of Corsican citron is quite diff erent from that of other Citrus species.Bergamot, grapefruit, mandarin, lemon, and orange   juices are dominated by naringin, neohesperidin, and neoeriocitrin as neohesperidoside forms and by hesperidin, narirutin, and didymin as rutinoside forms (34).

Conclusions
Volatile fractions of Corsican citron liqueur were dominated by limonene and γ-terpinene in accordance with the essential oil composition of C. medica var.Corsican peel.High proportions of rutin and neohesperidin characterized the fl avonoid fraction.The essential oil and polyphenolic composition of C. medica peel showed quantitative diff erences depending on fruit maturity.These results can be used for the quality assessment of organoleptic properties of food products (beverage, jam, nougat, and candy) derived from C. medica var.Corsican.Indeed, the commercial liqueur is traditionally made with mature fruits (harvested from September to November); however, the content of terpenic (γ-terpinene) and polyphenolic (diosmin, neohesperidin, casticin) compounds varied substantially during fruit development.

Table 2 .
Growing degree-days (GDD) and phenotypic characteristics of C. medica var.Corsican according to fruit development

Table 3 .
Volatile composition of the essential oil and commercial liqueur from C. medica