# Volatile and Flavonoid composition of the peel of Citrus medica L. var. Corsican fruit for quality assessment of its liqueur

N. Venturini, T. Barboni, F. Curk, J. Costa, J. Paolini

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N. Venturini, T. Barboni, F. Curk, J. Costa, J. Paolini. Volatile and Flavonoid composition of the peel of Citrus medica L. var. Corsican fruit for quality assessment of its liqueur. Food Technology and Biotechnology, 2014, 52 (4), pp.403-410. 10.17113/ftb.52.04.14.3717. hal-01147657

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

Nicolas Venturini<fnref n="1" />, Toussaint Barboni<fnref n="1" />, Franck Curk<fnref n="2" />, Jean Costa<fnref n="1" /> and Julien Paolini<fnref n="1" />*

Received: May 7, 2014

Accepted: October 6, 2014

## Summary

The volatile and flavonoid compositions of the peel of *Citrus medica* L. var. *Corsican* fruits cultivated in Corsica were studied according to the maturity of the citron fruits measured using growing degree-days. Quantitative variation with the stage of development of the fruit was observed using gas chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry/mass spectrometry. Thirty volatile compounds were identified in the peel essential oil. Limonene and γ-terpinene were the major compounds. The volatile compositions of commercial citron liqueurs were also characterized by high amounts of monoterpene hydrocarbons with the same two major components. The main flavonoid components of citron fruits and derived liqueurs were rutin and neohesperidin. This chemical characterization can be used for quality assessment of food products from *C. medica* var. *Corsican*.

**Key words:** *Citrus medica* L. var. *Corsican*, citrus peel, flavonoid compounds, essential oil, commercial citron liqueur

## 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 (2–6). 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 first *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 flowers 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 attributed to its external vesicles, which contain es-

<footnote n="1">¹University of Corsica, CNRS-UMR 6134, Laboratory of Natural Product Chemistry, BP 52, FR-20250 Corte, France</footnote>

<footnote n="2">²UR-INRA GEQA 110, Center INRA of Corsica, FR-20230 San Ghjulianu, France</footnote>

<footnote>*Corresponding author: Phone: +33 4 9545 0187; Fax: +33 4 9545 0257; E-mail: paolini@univ-corse.fr</footnote>
sential oils, rather than its pulp, which is seldom con-
sumed. Its skin is thick, and its white inner tissue, called
albedo, is consumed usually after 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 usual-
ly 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* (9–14). These studies showed quantitative
differences with regard to the major constituents: limo-
nene, γ-terpinene, geranial and neral. Thus, several chem-
ical 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, ner-
al, geranial, nerol and geraniol), the citron cultivars were
classified in four main oil chemotypes. To our knowledge,
there have been no studies on the polyphenolic composi-
tion of *C. medica* var. *Corsican*. However, some articles
have dealt with flavonoid components in other varieties of
*C. medica* (16–18). The flavonoids reported in the fruits are
rutin and hesperidin derivatives (sakuranetin, 7-O-meth-
yl-aromadendrin, dihydrokaempferide). High amounts of
glycosylated flavones, flavanones, and dihydrochalcone
were also identified; the major compounds were C- and/or
O-glycosides of apigenin, phloretin, diosmetin and hes-
peretin.

As part of our investigations on the chemical charac-
terization of citron varieties (15) and alcoholic beverages
from Corsica (19), the aim of this work is to provide a bet-
ter knowledge of the chemical characteristics (volatile and
polyphenolic compositions) of typical commercial liqueurs
from *C. medica* var. *Corsican*. This characterization should
lead to a better valorization of these products by standard-
ization of their quality.

Materials and Methods

Sampling of plant material

One thousand and five 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, Mediterra-
nean climate, soil derived from alluvial deposits and clas-
sified as fersiallitic). Citron fruits were collected each
month from July to January. Phenotypic characteristics of
fruits such as mass, length, width, and colour were mea-
sured 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 evalu-
ated 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 inde-
pendent of interannual variations of temperature, but de-
pendent on the species or varieties and environmental fac-
tors. The flowers of *C. medica* var. *Corsican* were scored at
the date of anthesis (flowers blooming on April 15, 2010).
From this date until the final harvest of the fruit (January
4, 2011), the air temperature was measured daily by re-
corders placed on the canopy of three trees (facing east).

Isolation of essential oil

The peel of *C. medica* L. var. *Corsican* was water dis-
tilled (5 h) using a Clevenger-type apparatus (Midiscienc-
es, Fuveau, France) according to the method recommend-
ed 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 filtered on a Büchner funnel and extracted with ethyl acetate (3×100 mL). The solution was evaporated, recovered in methanol (50 mL) and filtered 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 appa-
ratus equipped with dual flame ionization detection (FID)
systems and two fused silica capillary columns (60 m×0.22
mm i.d.; film thickness 0.25 µm) coated with Rtx-1 (poly-
dimethylsiloxane) 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 capil-
lary 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 identification of individual vola-
tiles was based on comparison of calculated retention in-
dices (apolar and polar columns) and mass spectra with
those of our own library of authentic compounds or litera-
ture data (I) (21). Retention indices (I and I, respectively)
of the compounds were determined relative to the reten-
tion times of series of *n*-alkanes (C~5~–C~30) with linear inter-
polation, using the Van den Dool and Kratz equation (22)
and software from Perkin-Elmer. The relative concentra-
tions 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 (NH₄OAc, LC-MS grade) obtained from Fisher Scientific (Illkirch, France). Deionized water was purified using a MilliQ water (Millipore, Bedford, MA, USA) purification system. Reference flavonoids (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 filtered through a 0.2-µm polytetrafluoroethylene (PTFE) filter. Direct-infusion MS analyses were performed with reference solutions at a concentration of 0.1 mg/mL in volume ratio of ACN/H₂O=5:5 and 0.1 % NH₄OH. 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-perfomance 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 (Pheromex) 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) NH₄AcO acetate buffer at a flow 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 (Tor-
ronto, Canada) 3200 QTRAP linear triple quadrupole fit-
ted 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 settings 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 tempera-
ture 550 °C. The following settings 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) ex-
periments, the MS parameters were set as follows in posi-
tive ion mode: declustering potential (DP) 50 V, entrance
potential (EP) 10 V, collision energy (CE) –35 V, and colli-
sion energy spread (CES) ±15 V, and in negative mode: DP
–80 V, EP –10 V, CE –35 V, and CES ±15 V. The software
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

(flow rate: 10 µL/min) using the automated component
optimization function of the Analyst software. 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
identification was allowed by comparison of retention
time, MRM transition and the EPI mass spectrum of refer-
ence compounds. An external standard method was used
for quantification of flavonoid compounds. The quantifi-
cation was performed using a calibration curve obtained
by serial diluted reference solution (six levels of concen-
trations with three injections per level) of each compound
in our chromatographic conditions.

Identification of flavonoid components

The optimized MS/MS parameters (declustering po-
tential, entrance potential, collision cell entrance potential,
collision energy, collision cell, exit potential) and MRM
transitions for each flavonoid component of *C. medica* var.
*Corsican* extract were described in Table 1. According to
the data previously reported in literature (23–26), the frag-
mentation 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) (fibre 2 cm, 30 µm thickness) 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 after three different experiments at 20, 50 and 70 °C, respectively, for each different 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. After sampling, the SPME fibre 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 fibre was reconditioned for 5 min in the GC injection port at 260 °C. HS-SPME and subsequent analyses were performed in triplicate. Volatile and flavonoid analyses of commercial citron liqueurs were performed using the same experimental parameters as those described for essential oil and solvent extractions, respectively.

**Results and Discussion**

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-
<table><caption>Table 1. MS/MS detection parameters of 13 flavonoid components of *Citrus medica* var. *Corsican*</caption><thead><tr><th rowspan="2">Elution order</th><th rowspan="2">Compound</th><th rowspan="2">Type</th><th rowspan="2">t<br>min</th><th colspan="2">Transition/(m/z)*</th><th colspan="5">Optimized MS parameters</th></tr><tr><th>Q1 mass<br>Da</th><th>Q3 mass<br>Da</th><th>DP<br>V</th><th>EP<br>V</th><th>CEP<br>V</th><th>CE<br>V</th><th>CXP<br>V</th></tr></thead><tbody><tr><td>1</td><td>Flavanomarein</td><td>monoglycosylated<br>flavanone</td><td>6.7</td><td>449.1</td><td>287.1</td><td>-70</td><td>-10</td><td>-28</td><td>-50</td><td>-3</td></tr><tr><td>2</td><td>Hyperoside</td><td>monoglycosylated<br>flavonol</td><td>8.1</td><td>463.0</td><td>300.1</td><td>-105</td><td>-10</td><td>-18</td><td>-28</td><td>-4</td></tr><tr><td>3</td><td>Narirutin</td><td>diglycosylated<br>flavanone</td><td>8.5</td><td>579.1</td><td>270.4</td><td>-80</td><td>-8.5</td><td>-32</td><td>-30</td><td>-4</td></tr><tr><td>4</td><td>Diosmin</td><td>diglycosylated<br>flavone</td><td>7.5</td><td>607.1</td><td>299.1</td><td>-80</td><td>-10</td><td>-34</td><td>-35</td><td>-3</td></tr><tr><td>5</td><td>Neohesperidin</td><td>diglycosylated<br>flavanone</td><td>7.5</td><td>609.1</td><td>301.1</td><td>-80</td><td>-10</td><td>-34</td><td>-35</td><td>-3</td></tr><tr><td>6</td><td>Rutin</td><td>diglycosylated<br>flavonol</td><td>7.6</td><td>609.1</td><td>300.1</td><td>-80</td><td>-10</td><td>-34</td><td>-35</td><td>-3</td></tr><tr><td>7</td><td>Retusin</td><td>polymethoxylated<br>flavonol</td><td>15.5</td><td>357.1</td><td>299.3</td><td>-45</td><td>-9</td><td>-24</td><td>-20</td><td>-10</td></tr><tr><td>8</td><td>Robinetin<br>trimethylether</td><td>polymethoxylated<br>flavonol</td><td>7.7</td><td>343.1</td><td>313.1</td><td>-70</td><td>-10</td><td>-24</td><td>-40</td><td>-3</td></tr><tr><td>9</td><td>Luteolin</td><td>flavone</td><td>9.9</td><td>287.0</td><td>153.1</td><td>20</td><td>10</td><td>17</td><td>35</td><td>6</td></tr><tr><td>10</td><td>Sinensetin</td><td>polymethoxylated<br>flavonone</td><td>9.7</td><td>373.1</td><td>343.3</td><td>71</td><td>4.5</td><td>30</td><td>27</td><td>6</td></tr><tr><td>11</td><td>Casticin</td><td>polymethoxylated<br>flavonol</td><td>10.1</td><td>375.1</td><td>359.1</td><td>50</td><td>10</td><td>20</td><td>35</td><td>3</td></tr><tr><td>12</td><td>Nobiletin</td><td>polymethoxylated<br>flavone</td><td>13.4</td><td>403.1</td><td>373.2</td><td>56</td><td>4.5</td><td>16</td><td>27</td><td>6</td></tr><tr><td>13</td><td>Tangeretin</td><td>polymethoxylated<br>flavone</td><td>14.3</td><td>373.1</td><td>343.3</td><td>81</td><td>8.5</td><td>28</td><td>25</td><td>14</td></tr></tbody></table>

DP=declustering potential, EP=entrance potential, CEP=collision cell entrance potential, CE=collission energy, CXP=collision cell exit potential

*MRM=Q1 [molecular ions] and Q3 [fragment ions]
Flavanomarein: Q1 [M-H]⁻ / Q3 [Y0]⁻ = [M-C₆H₁₁O₅]⁻
Hyperoside: Q1 [M-H]⁻ / Q3 [Y0-H]⁻ = [M-C₆H₁₂O₅]⁻
Narirutin: Q1 [M-H]⁻ / Q3 [Y0-H]⁻ = [M-C₁₂H₂₂O₈]⁻
Diosmin: Q1 [M-H]⁻ / Q3 [Y0]⁻ = [M-C₁₂H₂₁O₈]⁻
Neohesperidin: Q1 [M-H]⁻ / Q3 [Y0]⁻ = [M-C₁₂H₂₁O₈]⁻
Rutin: Q1 [M-H]⁻ / Q3 [Y0-H]⁻ = [M-C₁₂H₂₂O₈]⁻

Retusin: Q1 [M-H]⁻ / Q3 [M-H-2CH₃-CO]⁻
Robinetin trimethylether: Q1 [M-H]⁻ / Q3 [M-H-2CH₃]⁻
Luteolin: Q1 [M+H]⁺ / Q3 [1,3A]⁺ = [M+H-C₆H₆O₂]⁺
Sinensetin: Q1 [M+H]⁺ / Q3 [M+H-2CH₃]⁺
Casticin: Q1 [M+H]⁺ / Q3 [M-CH₃]⁺
Nobiletin: Q1 [M+H]⁺ / Q3 [M+H-2CH₃]⁺
Tangeretin: Q1 [M+H]⁺ / Q3 [M+H-2CH₃]⁺

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 difference of climatic factors between Corsica (Mediterranean area) and Brazil (tropical area) or by the difference 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, after 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. After 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 12.5–13.5 cm.

## 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 identified in the peel essential oil (97.6–99.5 % of the total composition). Twelve monoterpenes hydrocarbons, twelve oxygenated monoterpenes, four sesquiterpene hydrocarbons, and two linear oxygenated components were reported. Essential oil was dominated by monoterpenes hydrocarbons (66.8–82.5 %). Limonene was the major compound (54.2–60.6 %) followed by
<table><caption>Table 2. Growing degree-days (GDD) and phenotypic characteristics of *C. medica* var. *Corsican* according to fruit development</caption><thead><tr><th></th><th>April</th><th>May</th><th>June</th><th>July</th><th>August</th><th>September</th><th>October</th><th>November</th><th>December</th><th>January</th></tr></thead><tbody><tr><td>Formula A*</td><td>–</td><td>15</td><td>25</td><td>17</td><td>27</td><td>24</td><td>14</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Formula B*</td><td>15</td><td>14</td><td>–</td><td>–</td><td>–</td><td>–</td><td>17</td><td>30</td><td>31</td><td>–</td></tr><tr><td>Formula C*</td><td>–</td><td>2</td><td>5</td><td>14</td><td>4</td><td>6</td><td>–</td><td>–</td><td>–</td><td>–</td></tr><tr><td>Monthly GDD</td><td>68.2</td><td>300.1</td><td>336.7</td><td>444.9</td><td>434.1</td><td>343.7</td><td>177.8</td><td>67.8</td><td>31.6</td><td>–</td></tr><tr><td>Total GDD</td><td>–</td><td>–</td><td><strong>705.0</strong></td><td><strong>1149.9</strong></td><td><strong>1584.0</strong></td><td><strong>1927.7</strong></td><td><strong>2105.5</strong></td><td><strong>2173.3</strong></td><td><strong>2204.9</strong></td><td>–</td></tr><tr><td>Mass/g</td><td>–</td><td>–</td><td>–</td><td>119.1±33.5</td><td>349.2±62.7</td><td>821.4±201.1</td><td>1218.6±187.1</td><td>1122.6±272.2</td><td>1203.0±146.3</td><td>1289.1±281.7</td></tr><tr><td>Lenght/cm</td><td>–</td><td>–</td><td>–</td><td>6.9±0.8</td><td>9.3±1.0</td><td>12.0±1.8</td><td>12.4±1.5</td><td>13.0±1.0</td><td>12.4±1.3</td><td>12.2±1.1</td></tr><tr><td>Width/cm</td><td>–</td><td>–</td><td>–</td><td>6.0±0.6</td><td>9.1±0.7</td><td>11.7±1.4</td><td>13.5±1.1</td><td>12.5±1.3</td><td>13.3±0.5</td><td>13.3±1.2</td></tr><tr><td>Colour</td><td>–</td><td>–</td><td>–</td><td>dark green</td><td>dark green</td><td>green-yellow</td><td>green-yellow</td><td>pale yellow</td><td>yellow</td><td>yellow-orange</td></tr></tbody></table>

γ-terpinene (6.7–15.2 %). The oxygenated monoterpenes nerol, neral, geraniol and geranial were present in relatively high concentrations (2.9–8.2, 2.1–6.6, 1.8–5.9, and 2.1–5.7 %, respectively). These results are in accordance with those of Lota *et al.* (13) and Venturini *et al.* (15). Indeed, these authors have reported a chemotype limonene/γ-terpinene of the Corsican citron variety. This combination had previously been observed in various *Citrus* species including limes (*C. latifolia* and *C. aurantifolia*), tangerine (*C. reticulata* × *C. sinensis*), mandarin (*C. reticulata* Blanco) and lemon (*C. limon*) (27–29).

During fruit development, the yield and the chromatographic profile of essential oil exhibited significant differences 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 profile dominated by limonene (57.8–54.6 %) and γ-terpinene (11.5–15.2 %). The content of the major component, limonene, can be considered stable (54.2 % in July, 54.6 % in January). A significant difference between immature and mature fruits was only observed in the content of γ-terpinene. 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 difference in the contents of oxygenated compounds.

## Analysis of flavonoid compounds

As far as we know, the flavonoid composition of *C. medica* var. *Corsican* species has not been reported previously. Thirteen compounds were identified including three glycosylated flavanones, two glycosylated flavonols, one glycosylated flavone, one aglycone flavone, three polymethoxylated flavonols and three polymethoxylated flavones (Table 4). The main chemical classes determined on dry mass basis were the glycosylated flavonols (10.0–14.3 mg/g) followed by glycosylated flavanones (1.1–10.2 mg/g) and flavones (0.5–9.9 mg/g).

The main components were identified as rutin, diosmin, and neohesperidin. Mass fractions of flavonoids changed differently 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 flavonoids 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 flavonoid 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 flavanones, neohesperidosides and rutinosides, had previously been reported in the peel compositions of other *Citrus* species (33). These two forms were identified in *C. medica* extract with neohesperidin and rutin as major components. However, the flavonoid composition of Corsican citron is quite different from that of other *Citrus* species. Bergamot, grapefruit, mandarin, lemon, and orange

<footnote>*according to Stenzel *et al.* (21)</footnote>
<table><caption>Table 3. Volatile composition of the essential oil and commercial liqueur from *C. medica* var. *Corsican* peel</caption><thead><tr><th rowspan="2">Elution order</th><th rowspan="2">Compound</th><th rowspan="2">I<sub>1</sub></th><th rowspan="2">I<sub>a</sub></th><th rowspan="2">I<sub>p</sub></th><th colspan="7">Y(essential oil)/%</th><th rowspan="2">φ(component<br>in liqueur)*</th></tr><tr><th>0.40</th><th>0.62</th><th>0.70</th><th>φ(component in essential oil)/%</th><th>0.65</th><th>0.72</th><th>0.69</th><th>%</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th>July</th><th>August</th><th>September</th><th>October</th><th>November</th><th>December</th><th>January</th><th></th><th></th></tr></thead><tbody><tr><td>1</td><td>α-Thujene</td><td>932</td><td>922</td><td>1023</td><td>0.1±0.1</td><td>0.2±0.1</td><td>0.3±0.0</td><td>0.3±0.0</td><td>0.3±0.1</td><td>0.4±0.1</td><td>0.4±0.1</td><td>1.2±0.3</td></tr><tr><td>2</td><td>α-Pinene</td><td>936</td><td>931</td><td>1022</td><td>0.4±0.2</td><td>0.7±0.1</td><td>0.9±0.1</td><td>1.0±0.1</td><td>0.9±0.4</td><td>1.1±0.3</td><td>1.0±0.3</td><td>3.6±0.5</td></tr><tr><td>3</td><td>6-Methylhept-5-en-2-one</td><td>963</td><td>963</td><td>1337</td><td>0.1±0.0</td><td>0.1±0.0</td><td>0.1±0.0</td><td>tr</td><td>tr</td><td>tr</td><td>0.2±0.1</td><td>–</td></tr><tr><td>4</td><td>Sabinene</td><td>964</td><td>969</td><td>1111</td><td>0.1±0.1</td><td>0.1±0.1</td><td>0.2±0.0</td><td>0.2±0.0</td><td>0.2±0.1</td><td>0.2±0.1</td><td>0.2±0.1</td><td>0.3±0.2</td></tr><tr><td>5</td><td>β-Pinene</td><td>978</td><td>970</td><td>1110</td><td>0.4±0.2</td><td>0.7±0.1</td><td>0.8±0.1</td><td>0.9±0.0</td><td>0.9±0.3</td><td>1.0±0.2</td><td>1.0±0.2</td><td>2.8±0.3</td></tr><tr><td>6</td><td>Myrcene</td><td>987</td><td>979</td><td>1159</td><td>1.1±0.3</td><td>1.4±0.2</td><td>1.6±0.1</td><td>1.6±0.1</td><td>1.4±0.3</td><td>1.5±0.2</td><td>1.3±0.1</td><td>2.4±0.2</td></tr><tr><td>7</td><td>α-Terpinene</td><td>1013</td><td>1008</td><td>1178</td><td>0.1±0.1</td><td>0.2±0.0</td><td>0.3±0.0</td><td>0.3±0.0</td><td>0.3±0.1</td><td>0.2±0.1</td><td>0.3±0.1</td><td>–</td></tr><tr><td>8</td><td>p-Cymene</td><td>1015</td><td>1010</td><td>1169</td><td>2.4±1.1</td><td>1.4±0.7</td><td>0.5±0.1</td><td>0.7±0.4</td><td>0.6±0.3</td><td>2.0±0.6</td><td>1.7±0.5</td><td>1.3±0.2</td></tr><tr><td>9</td><td>Limonene</td><td>1025</td><td>1020</td><td>1199</td><td>54.2±4.0</td><td>57.8±1.7</td><td>60.3±1.0</td><td>60.6±1.2</td><td>56.1±1.9</td><td>57.9±3.0</td><td>54.6±1.1</td><td>60.5±1.3</td></tr><tr><td>10</td><td>(Z)-β-Ocimene</td><td>1029</td><td>1026</td><td>1230</td><td>0.2±0.1</td><td>0.3±0.1</td><td>0.2±0.0</td><td>0.2±0.0</td><td>0.1±0.1</td><td>0.2±0.1</td><td>0.2±0.0</td><td>–</td></tr><tr><td>11</td><td>(E)-β-Ocimene</td><td>1041</td><td>1043</td><td>1251</td><td>0.7±0.1</td><td>1.2±0.2</td><td>1.2±0.1</td><td>1.6±0.2</td><td>1.4±0.3</td><td>1.8±0.3</td><td>2.0±0.2</td><td>–</td></tr><tr><td>12</td><td>γ-Terpinene</td><td>1051</td><td>1047</td><td>1243</td><td>6.7±2.6</td><td>11.5±0.2</td><td>12.5±0.4</td><td>14.4±0.8</td><td>14.2±2.4</td><td>13.6±1.7</td><td>15.2±1.4</td><td>21.8±1.0</td></tr><tr><td>13</td><td>Terpinolene</td><td>1082</td><td>1078</td><td>1280</td><td>0.4±0.1</td><td>0.6±0.1</td><td>0.6±0.0</td><td>0.6±0.1</td><td>0.7±0.1</td><td>0.6±0.1</td><td>0.7±0.1</td><td>1.0±0.3</td></tr><tr><td>14</td><td>Nonanal</td><td>1084</td><td>1083</td><td>1388</td><td>0.1±0.1</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>–</td></tr><tr><td>15</td><td>Linalool</td><td>1086</td><td>1085</td><td>1544</td><td>0.6±0.1</td><td>0.5±0.1</td><td>0.4±0.1</td><td>0.3±0.1</td><td>0.5±0.1</td><td>0.4±0.2</td><td>0.5±0.2</td><td>–</td></tr><tr><td>16</td><td>Citronellal</td><td>1129</td><td>1132</td><td>1479</td><td>0.1±0.1</td><td>0.1±0.0</td><td>0.2±0.1</td><td>0.1±0.0</td><td>0.1±0.1</td><td>0.2±0.1</td><td>0.5±0.1</td><td>–</td></tr><tr><td>17</td><td>Isoneral</td><td>1140</td><td>1142</td><td>1556</td><td>0.5±0.1</td><td>0.3±0.1</td><td>0.4±0.1</td><td>0.2±0.0</td><td>0.3±0.2</td><td>0.1±0.0</td><td>0.1±0.1</td><td>–</td></tr><tr><td>18</td><td>Isogeranial</td><td>1156</td><td>1159</td><td>1748</td><td>0.8±0.2</td><td>0.5±0.1</td><td>0.7±0.1</td><td>0.4±0.1</td><td>0.6±0.3</td><td>0.2±0.0</td><td>0.3±0.1</td><td>–</td></tr><tr><td>19</td><td>Terpinen-4-ol</td><td>1164</td><td>1161</td><td>1600</td><td>0.4±0.1</td><td>0.3±0.1</td><td>0.2±0.0</td><td>0.2±0.1</td><td>0.3±0.1</td><td>0.2±0.1</td><td>0.2±0.1</td><td>–</td></tr><tr><td>20</td><td>α-Terpineol</td><td>1176</td><td>1179</td><td>1700</td><td>0.9±0.2</td><td>0.6±0.1</td><td>0.6±0.0</td><td>0.5±0.1</td><td>0.6±0.3</td><td>0.4±0.2</td><td>0.7±0.3</td><td>–</td></tr><tr><td>21</td><td>Nerol</td><td>1210</td><td>1212</td><td>1799</td><td>5.9±0.7</td><td>4.8±0.3</td><td>1.8±0.1</td><td>3.9±1.0</td><td>4.3±1.0</td><td>2.5±1.9</td><td>2.6±0.9</td><td>–</td></tr><tr><td>22</td><td>Neral</td><td>1215</td><td>1214</td><td>1679</td><td>5.7±1.5</td><td>3.8±0.5</td><td>4.5±0.4</td><td>2.1±0.1</td><td>3.3±1.5</td><td>3.6±0.8</td><td>4.7±0.6</td><td>0.1±0.1</td></tr><tr><td>23</td><td>Geraniol</td><td>1235</td><td>1233</td><td>1843</td><td>6.6±1.3</td><td>5.8±0.4</td><td>2.1±0.2</td><td>5.1±0.9</td><td>5.4±0.5</td><td>2.7±1.9</td><td>2.3±0.6</td><td>–</td></tr><tr><td>24</td><td>Geranial</td><td>1244</td><td>1245</td><td>1710</td><td>8.2±2.3</td><td>5.1±0.7</td><td>6.6±0.6</td><td>2.9±0.2</td><td>4.2±1.3</td><td>4.9±1.0</td><td>6.6±1.1</td><td>0.1±0.1</td></tr><tr><td>25</td><td>Neryl acetate</td><td>1342</td><td>1343</td><td>1725</td><td>0.3±0.2</td><td>0.2±0.1</td><td>0.5±0.2</td><td>0.3±0.1</td><td>0.5±0.2</td><td>0.8±0.2</td><td>0.6±0.3</td><td>–</td></tr><tr><td>26</td><td>Geranyl acetate</td><td>1362</td><td>1357</td><td>1752</td><td>0.4±0.2</td><td>0.3±0.1</td><td>0.3±0.2</td><td>0.2±0.1</td><td>0.4±0.1</td><td>0.4±0.1</td><td>0.5±0.2</td><td>tr</td></tr><tr><td>27</td><td>(E)-β-Caryophyllene</td><td>1421</td><td>1424</td><td>1591</td><td>0.2±0.1</td><td>0.2±0.0</td><td>0.2±0.0</td><td>0.1±0.1</td><td>0.2±0.1</td><td>0.1±0.0</td><td>0.1±0.0</td><td>tr</td></tr><tr><td>28</td><td>trans-α-Bergamotene</td><td>1429</td><td>1431</td><td>1581</td><td>0.1±0.0</td><td>0.1±0.0</td><td>0.2±0.1</td><td>0.2±0.1</td><td>0.2±0.0</td><td>0.2±0.1</td><td>0.2±0.0</td><td>0.3±0.1</td></tr><tr><td>29</td><td>Germacrene D</td><td>1479</td><td>1475</td><td>1704</td><td>0.1±0.0</td><td>0.2±0.1</td><td>0.1±0.1</td><td>0.1±0.0</td><td>0.1±0.1</td><td>0.1±0.0</td><td>0.1±0.0</td><td>0.3±0.2</td></tr><tr><td>30</td><td>β-Bisabolene</td><td>1498</td><td>1502</td><td>1720</td><td>0.2±0.1</td><td>0.2±0.0</td><td>0.3±0.1</td><td>0.2±0.1</td><td>0.3±0.1</td><td>0.2±0.1</td><td>0.2±0.1</td><td>0.2±0.2</td></tr><tr><td colspan="2">Total</td><td>98.0±1.2</td><td>99.1±0.9</td><td>98.8±0.9</td><td>99.5±0.2</td><td>98.1±1.2</td><td>97.6±1.7</td><td>99.3±0.9</td><td>98.9±0.9</td><td></td><td></td><td></td></tr></tbody></table>

Order of elution is given on apolar column (Rtx-1)

I₁=Retention indices in the literature on the apolar column (21)

I₂=Retention indices on the apolar Rtx-1 column

I₃=Retention indices on the polar Rtx-Wax column

*N=9; mean values of three analyses of each three commercial samples obtained for maximum sum of total area±standard deviation

tr=trace (<0.05 %)

—not detected
<table><caption>Table 4. Flavonoid composition of solvent extract and commercial liqueur on dry mass basis from *C. medica* var. *Corsican* peel</caption><thead><tr><th rowspan="2">Elution order</th><th rowspan="2">Compound</th><th colspan="8">Solvent extract</th></tr><tr><th>July</th><th>August</th><th>September</th><th>October</th><th>November</th><th>December</th><th>January</th><th>Liqueur</th></tr><tr><td></td><td></td><td colspan="8">w(flavonoid)/(mg/g)</td></tr></thead><tbody><tr><td>1</td><td>Flavanomarein</td><td>1.4±0.3</td><td>1.3±0.3</td><td>1.1±0.3</td><td>0.9±0.3</td><td>1.0±0.2</td><td>1.3±0.3</td><td>1.3±0.3</td><td>1.1±0.3</td></tr><tr><td>2</td><td>Hyperosid</td><td>1.4±0.1</td><td>1.4±0.1</td><td>2.0±0.2</td><td>2.2±0.5</td><td>2.2±0.5</td><td>3.1±0.4</td><td>3.1±0.2</td><td>3.2±0.8</td></tr><tr><td>3</td><td>Narirutin</td><td>0.1±0.1</td><td>tr</td><td>0.1±0.0</td><td>0.1±0.0</td><td>0.1±0.0</td><td>0.1±0.1</td><td>0.1±0.1</td><td>tr</td></tr><tr><td>4</td><td>Diosmin</td><td>9.9±1.2</td><td>6.1±1.1</td><td>2.1±0.4</td><td>1.0±0.3</td><td>0.8±0.3</td><td>0.7±0.2</td><td>0.5±0.2</td><td>0.4±0.1</td></tr><tr><td>5</td><td>Neohesperidin</td><td>1.2±0.1</td><td>0.1±0.1</td><td>tr</td><td>5.4±1.0</td><td>6.9±0.8</td><td>8.7±0.9</td><td>8.7±0.6</td><td>4.5±0.5</td></tr><tr><td>6</td><td>Rutin</td><td>10.4±0.9</td><td>8.6±1.0</td><td>8.0±0.5</td><td>8.4±0.6</td><td>9.0±1.0</td><td>10.3±1.1</td><td>11.2±1.0</td><td>16.8±1.3</td></tr><tr><td>7</td><td>Retusin</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>-</td><td>tr</td></tr><tr><td>8</td><td>Robinetin trimethylether</td><td>0.4±0.2</td><td>0.2±0.0</td><td>0.6±0.2</td><td>0.4±0.2</td><td>0.2±0.1</td><td>0.5±0.1</td><td>0.3±0.1</td><td>0.3±0.2</td></tr><tr><td>9</td><td>Luteolin</td><td>0.1±0.0</td><td>0.1±0.1</td><td>tr</td><td>0.1±0.1</td><td>0.1±0.0</td><td>0.1±0.1</td><td>tr</td><td>tr</td></tr><tr><td>10</td><td>Sinensetin</td><td>tr</td><td>-</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td></tr><tr><td>11</td><td>Casticine</td><td>1.7±0.4</td><td>0.3±0.1</td><td>0.1±0.1</td><td>0.1±0.1</td><td>0.1±0.0</td><td>0.2±0.1</td><td>0.1±0.0</td><td>tr</td></tr><tr><td>12</td><td>Nobiletin</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td></tr><tr><td>13</td><td>Tangeretin</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>tr</td><td>-</td><td>-</td><td>tr</td></tr><tr><td colspan="2">Polymethoxylated flavones</td><td>0.1±0.1</td><td>0.1±0.1</td><td>tr</td><td>0.1±0.2</td><td>0.1±0.1</td><td>0.1±0.1</td><td>tr</td><td>tr</td></tr><tr><td colspan="2">Flavones</td><td>9.9±1.2</td><td>6.1±1.1</td><td>2.1±0.4</td><td>1.0±0.3</td><td>0.8±0.3</td><td>0.7±0.2</td><td>0.5±0.2</td><td>0.4±0.2</td></tr><tr><td colspan="2">Glycosylated flavanones</td><td>2.7±0.5</td><td>1.4±0.4</td><td>1.1±0.3</td><td>6.4±1.2</td><td>7.9±1.0</td><td>10.1±1.3</td><td>10.2±0.8</td><td>5.7±1.6</td></tr><tr><td colspan="2">Polymethoxylated flavonols</td><td>2.1±0.5</td><td>0.5±0.1</td><td>0.6±0.2</td><td>0.5±0.2</td><td>0.3±0.1</td><td>0.7±0.1</td><td>0.4±0.1</td><td>0.3±0.2</td></tr><tr><td colspan="2">Glycosylated flavonols</td><td>11.8±0.9</td><td>10.0±1.0</td><td>10.0±0.7</td><td>10.6±0.9</td><td>11.2±1.4</td><td>13.4±1.5</td><td>14.3±1.2</td><td>20.0±2.1</td></tr><tr><td colspan="2">Total</td><td>26.5±4.3</td><td>18.0±3.2</td><td>13.8±3.2</td><td>18.6±3.9</td><td>20.3±3.1</td><td>25.0±3.9</td><td>25.3±3.7</td><td>26.4±3.2</td></tr></tbody></table>

Order of elution corresponds to that in Table 1

*N= 9, mean values of three analyses of three commercial samples±standard deviation tr=trace

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 flavonoid fraction. The essential oil and polyphenolic composition of *C. medica* peel showed quantitative differences 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 (dosmin, neohesperidin, casticin) compounds varied substantially during fruit development.

## Acknowledgements

The authors are indebted to the Territorial Collectivity of Corsica (CTC) and the Agency for the Economic Development of Corsica (ADEC) for partial financial support. The authors are also grateful to the Mavela distillery (Aleria, Corsica) for liqueur samples.

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