# Integrated Analysis by GC(RI), GC-MS and 13C NMR of Fortunella japonica Leaf Volatiles Obtained by Hydrodistillation, Microwave- assisted Hydrodistillation and Hydrolate Extraction

Sylvain Sutour, Luro François, Joseph Casanova, Félix Tomi

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Sylvain Sutour, Luro François, Joseph Casanova, Félix Tomi. Integrated Analysis by GC(RI), GC-MS and 13C NMR of Fortunella japonica Leaf Volatiles Obtained by Hydrodistillation, Microwave-assisted Hydrodistillation and Hydrolate Extraction. Natural Product Communications, 2017, 12 (3), pp.431-434. 10.1177/1934578X1701200331. hal-02127464

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Distributed under a Creative Commons Attribution 4.0 International License
Integrated Analysis by GC(RI), GC-MS and ¹³C NMR of
*Fortunella japonica* Leaf Volatiles Obtained by Hydrodistillation, Microwave-assisted Hydrodistillation and Hydrolate Extraction

Sylvain Sutour<sup>a</sup>, François Luro<sup>b</sup>, Joseph Casanova<sup>a</sup> and Félix Tomi<sup>a,*</sup>

felix.tomi@univ-corse.fr

Received: June 22<sup>nd</sup>, 2016; Accepted: December 4<sup>th</sup>, 2016

The chemical composition of the essential oil (EO), microwave extract (ME) and hydrolate extract (HE) from the same batch of leaves of *Fortunella japonica*, was investigated by combination of chromatographic (GC, CC) and spectroscopic techniques (GC-MS, ¹³C NMR). *F. japonica* essential oil and extracts are complex mixtures of 28-60 compounds being mainly oxygenated sesquiterpenes. The EO composition was dominated by germacrene D (14.9%), β-elemol (9.1%), *cis*-guai-6-en-10β-ol (6.3%), β-eudesmol (5.5%), and δ-elemene (5.2%). Limonene was the unique monoterpene identified at appreciable amount (7.1%). The extract obtained by microwave assisted hydrodistillation contained as main components: β-elemol (12.4%), germacrene D (9.9%), *cis*-guai-6-en-10β-ol (9.0%), β-eudesmol (8.2%), germacra-1(10),5-dien-4α-ol (7.1%) and α-eudesmol (6.4%). Finally, the highest content of oxygenated sesquiterpenes (near 92%) was found in the hydrolate extract displaying cryptomeridiol (23.3%, but totally absent in the EO and ME), β-eudesmol (20.6%) and α-eudesmol (10.7%). Combined analysis by chromatographic and spectroscopic techniques appeared useful for identification of various sesquiterpenols bearing a tertiary alcohol function.

**Keywords:** *Fortunella japonica*, Essential oil, Microwave extract, Hydrolate extract.

The *Citrus* genus belongs to the plant family Rutaceae, which also includes *Poncirus* and *Fortunella*. Fruits from *Fortunella* species are called “kumquat”. Although when split they present the typical segmented appearance of a citrus fruit, kumquats are ovoid or oblong and smaller than fruits of *Citrus* species (for example, mandarins, oranges, lemons) [1]. The chemical composition of essential oils (EO) isolated from peel of *Fortunella* species has been substantially investigated and a summary of the results was reported in a previous paper [2]. In short, *Fortunella* peel oils contain mainly limonene (76.7%-87.1-96.9%), as do peel oils of *Citrus* species. In contrast, only a few papers have reported on the chemical composition of kumquat leaf oil. As soon as 1969, Scora *et al.* mentioned a leaf oil sample from *F. crassifolia* containing α-terpineol, beside other oxygenated monoterpenes [3]. Leaf oil of *F. margarita* of Chinese origin was dominated by linalool and terpenol beside methyl *N*-methylanthranilate [4]. In contrast, γ-eudesmol, β-elemol, and β-eudesmol were the most abundant compounds of Colombian *F. margarita* leaf oil [5]. Similarly, leaves of *F. margarita* grown in Egypt produced an essential oil that contained mainly β-eudesmol, α-murolene and β-gurjunene [6]. Major compounds of leaf oils from three *Fortunella* species of Greek origin were germacrene D and elemol [7].

Concerning *F. japonica* (Thunb.) Swing., the main components of the leaf oil from Nepal were the monoterpenes linalool (35.1%) and geraniol (12.7%), beside eugenol (14.8%) [8], while leaf oil from Thailand contained mainly β-pinene (47.4%), limonene (10.2%) and linalool (9.8%) [9]. In contrast, the main components of EO isolated from leaves of *F. japonica* grown in Vietnam were elemol (17.7%), β-eudesmol (16.6%) and epi-bicyclosesquiphellandrene (16.6%) [10]. EOs isolated from leaves of *F. japonica* cultivated in Egypt, harvested at different periods of the year, contained elemol (up to

19.4%), δ-cadinene (up to 16.0%), γ-eudesmol (up to 11.9%), guaiol (up to 11.7%), and τ-cadinol (up to 11.1%) [11]. Recently, our attention was focused on the identification, in the EOs of *Fortunella* species, of germacra-1(10),5-dien-4α-ol whose confusing spectral data and retention index values were reported in the literature [12]. In parallel, we reported the main components contained in the EOs as β-elemol (8.7-30.9%), germacrene D (2.1-16.3%), β-eudesmol (4.6-13.1%), eremophil-9-en-11-ol (1.4-11.2%), valerianol (1.4-8.9%) and α-eudesmol (3.1-8.8%).

Hydrodistillation (HD) is the most common approach to extract volatile compounds from medicinal herbs and plants. However, other extraction techniques have been applied to improve mainly extraction time, yield and sample preparation costs. They are mainly used to enrich extracts with valuable compounds. Among these techniques, microwave assisted hydrodistillation (MAHD), a green chemistry technique, was developed during the past decade and used for the extraction of volatiles from various aromatic plants [13]. In parallel, fragrance and cosmetic industries are more and more interested by water soluble compounds contained in the hydrolate. Indeed, these compounds can be easily incorporated into a cosmetic formulation and hydrolate is a large resource available simultaneously with the EO.

Therefore, the aim of the present work was to investigate the chemical compositions of the microwave extract (ME) and the hydrolate extract (HE) and to compare them with that of the EO obtained from the same batch of leaves of *Fortunella japonica*. Advantages and disadvantages of each mode of extraction will be discussed. Special attention will be brought to the identification of components (particularly sesquiterpenols) by using a combination of chromatographic and spectroscopic techniques.

<footnote><sup>a</sup>Université de Corse-CNRS, UMR 6134 SPE, Equipe Chimie et Biomasse, Route des Sanguinaires, 20000 Ajaccio, France</footnote>

<footnote><sup>b</sup>UMR 1390 AGAP, INRA, 20230 San Ghjulianu, France</footnote>
<table><caption>Table 1: Chemical composition of essential oil (EO), microwave extract (ME) and hydrolate extract (HE) from leaves of *Fortunella japonica*.</caption><thead><tr><th>N°</th><th>Components<sup>a</sup></th><th>RI<sup>b</sup></th><th>RI<sup>c</sup></th><th>HE</th><th>ME</th><th>HE</th><th>Identification</th></tr></thead><tbody><tr><td>1</td><td>α-Pinene</td><td>931</td><td>1022</td><td>tr</td><td>tr</td><td>0.1</td><td>GC(RI), MS</td></tr><tr><td>2</td><td>Sabinene</td><td>965</td><td>1124</td><td>tr</td><td>tr</td><td>-</td><td>GC(RI), MS</td></tr><tr><td>3</td><td>β-Pinene</td><td>971</td><td>1114</td><td>tr</td><td>tr</td><td>-</td><td>GC(RI), MS</td></tr><tr><td>4</td><td>Myrcene</td><td>980</td><td>1159</td><td>0.4</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>5</td><td>α-Phellandrene</td><td>994</td><td>1164</td><td>tr</td><td>-</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>6</td><td>3-Carene</td><td>1003</td><td>1148</td><td>0.1</td><td>-</td><td>0.4</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>7</td><td>β-Phellandrene*</td><td>1021</td><td>1210</td><td>tr</td><td>-</td><td>-</td><td>GC(RI), MS</td></tr><tr><td>8</td><td>Limonene*</td><td>1021</td><td>1217</td><td>7.1</td><td>0.1</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>9</td><td>(Z)-β-Ocimene</td><td>1023</td><td>1232</td><td>tr</td><td>-</td><td>-</td><td>GC(RI), MS</td></tr><tr><td>10</td><td>(E)-β-Ocimene</td><td>1036</td><td>1249</td><td>0.6</td><td>-</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>11</td><td>Terpinolene</td><td>1076</td><td>1280</td><td>tr</td><td>-</td><td>-</td><td>GC(RI), MS</td></tr><tr><td>12</td><td>Linalool</td><td>1080</td><td>1551</td><td>tr</td><td>-</td><td>0.4</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>13</td><td>δ-Elemene</td><td>1339</td><td>1467</td><td>5.2</td><td>2.1</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>14</td><td>α-Cubebene</td><td>1345</td><td>1454</td><td>0.1</td><td>tr</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>15</td><td>α-Ylangene</td><td>1368</td><td>1480</td><td>0.1</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>16</td><td>α-Copaene</td><td>1373</td><td>1488</td><td>0.1</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>17</td><td>β-Bourbonene</td><td>1381</td><td>1515</td><td>0.2</td><td>0.3</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>18</td><td>β-Elemene</td><td>1384</td><td>1588</td><td>3.3</td><td>3.3</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>19</td><td>β-Ylangene</td><td>1413</td><td>1568</td><td>0.3</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>20</td><td>(E)-β-Caryophyllene</td><td>1423</td><td>1593</td><td>1.6</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>21</td><td>β-Copaene</td><td>1425</td><td>1586</td><td>0.3</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>22</td><td>Thujopsene</td><td>1435</td><td>1618</td><td>0.4</td><td>tr</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>23</td><td>β-Gurjunene</td><td>1441</td><td>1593</td><td>0.4</td><td>0.4</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>24</td><td>(E)-β-Farnesene</td><td>1448</td><td>1635</td><td>0.7</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>25</td><td>α-Humulene</td><td>1454</td><td>1666</td><td>tr</td><td>tr</td><td>0.2</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>26</td><td>α-Amorphene*</td><td>1475</td><td>1686</td><td>0.5</td><td>1.0</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>27</td><td>β-Chamigrene*</td><td>1475</td><td>1703</td><td>0.1</td><td>0.2</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>28</td><td>Germacrene D*</td><td>1475</td><td>1712</td><td><b>14.9</b></td><td><b>9.9</b></td><td>0.1</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>29</td><td>β-Selinene*</td><td>1475</td><td>1714</td><td>0.8</td><td>0.3</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>30</td><td>trans-β-Bergamotene*</td><td>1475</td><td>1718</td><td>0.2</td><td>0.4</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>31</td><td>γ-Humulene*</td><td>1475</td><td>1721</td><td>0.5</td><td>0.3</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>32</td><td>γ-Amorphene</td><td>1493</td><td>1671</td><td>tr</td><td>-</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>33</td><td>Bicyclogermacrene</td><td>1496</td><td>1728</td><td>0.8</td><td>tr</td><td>0.2</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>34</td><td>α-Selinene</td><td>1496</td><td>1720</td><td>0.2</td><td>tr</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>35</td><td>δ-Amorphene</td><td>1500</td><td>1635</td><td>1.6</td><td>0.2</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>36</td><td>α-Bulnesene</td><td>1502</td><td>1711</td><td>0.2</td><td>0.3</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>37</td><td>δ-Cadinene</td><td>1510</td><td>1751</td><td>1.0</td><td>0.5</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>38</td><td>β-Elemol</td><td>1532</td><td>2094</td><td><b>9.1</b></td><td><b>12.4</b></td><td><b>11.1</b></td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>39</td><td>(E)-Nerolidol</td><td>1551</td><td>2049</td><td>1.1</td><td>2.1</td><td>tr</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>40</td><td>Germacrene B</td><td>1552</td><td>1833</td><td>2.4</td><td>4.7</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>41</td><td>cis-Guai-6-en-10β-ol</td><td>1575</td><td>1993</td><td>6.3</td><td>9.0</td><td>2.4</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>42</td><td>Amorph-4-en-10β-ol</td><td>1577</td><td>2083</td><td>tr</td><td>1.6</td><td>0.1</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>43</td><td>Guaiol</td><td>1580</td><td>2083</td><td>1.2</td><td>1.5</td><td>1.3</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>44</td><td>Eudesm-6-en-4α-ol</td><td>1604</td><td>2092</td><td>2.2</td><td>0.8</td><td>2.3</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>45</td><td>7-epi-γ-Eudesmol</td><td>1609</td><td>2036</td><td>0.4</td><td>0.2</td><td>1.0</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>46</td><td>Germacra-1(10),5-dien-4α-ol</td><td>1611</td><td>2188</td><td>2.4</td><td><b>7.1</b></td><td>0.2</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>47</td><td>trans-Guai-6-en-10β-ol</td><td>1612</td><td>2158</td><td>1.4</td><td>1.8</td><td>0.6</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>48</td><td>γ-Eudesmol*</td><td>1614</td><td>2178</td><td>1.4</td><td>0.8</td><td>4.4</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>49</td><td>Alismol*</td><td>1617</td><td>2270</td><td>1.2</td><td>3.5</td><td>1.8</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>50</td><td>Eremophil-9-11-ol</td><td>1623</td><td>2198</td><td>4.2</td><td>4.4</td><td>1.0</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>51</td><td>τ-Muurolol</td><td>1630</td><td>2200</td><td>0.1</td><td>1.5</td><td>6.7</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>52</td><td>τ-Cadinol</td><td>1630</td><td>2173</td><td>0.1</td><td>0.8</td><td>0.2</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>53</td><td>Amorph-4-en-10α-ol*</td><td>1641</td><td>2210</td><td>2.2</td><td>0.1</td><td>0.2</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>54</td><td>β-Eudesmol*</td><td>1641</td><td>2244</td><td>5.5</td><td>8.2</td><td><b>20.6</b></td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>55</td><td>α-Cadinol*</td><td>1641</td><td>2244</td><td>0.7</td><td>1.1</td><td>0.4</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>56</td><td>Valerianol</td><td>1643</td><td>2214</td><td>2.1</td><td>1.8</td><td>3.3</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>57</td><td>α-Eudesmol</td><td>1646</td><td>2238</td><td>4.4</td><td>6.4</td><td><b>10.7</b></td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>58</td><td>7-epi-α-Eudesmol</td><td>1649</td><td>2228</td><td>0.5</td><td>0.2</td><td>-</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>59</td><td>Eudesm-7(11)-en-4α-ol</td><td>1682</td><td>2310</td><td>0.4</td><td>0.5</td><td>0.2</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>60</td><td>Cryptomeridiol</td><td>1791</td><td>2598</td><td>-</td><td>-</td><td><b>23.3</b></td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td>61</td><td>(Z)-Phytol</td><td>2074</td><td>2545</td><td>-</td><td>0.2</td><td>-</td><td>GC(RI), <sup>13</sup>C NMR</td></tr><tr><td>62</td><td>(E)-Phytol</td><td>2104</td><td>2620</td><td>0.3</td><td>3.8</td><td>0.1</td><td>GC(RI), MS, <sup>13</sup>C NMR</td></tr><tr><td colspan="4">Total</td><td>91.6</td><td>93.8</td><td>93.3</td><td></td></tr></tbody></table>

**Strategy for analysis of F. japonica essential oil (EO), microwave extract (ME) and hydrolate extract (HE):** The EO and both extracts were analyzed by GC(RI), GC-MS and <sup>13</sup>C NMR following a computerized method developed in our laboratory [14]. Major components were identified by comparison of their spectral data (MS and/or NMR) with those of authentic compounds

compiled in our laboratory-made MS and <sup>13</sup>C NMR spectral data libraries. A few compounds, mainly monoterpenes present in low content, were identified by GC(RI) and GC-MS. In order to identify minor components, the EO and ME were submitted to column chromatography (CC) and all the fractions of CC were analyzed by GC(RI) and <sup>13</sup>C NMR. <sup>13</sup>C NMR confirmed the identification of

<footnote><sup>a</sup>Order of elution and percentages of individual components are given on an apolar column (BP-1), except those with an asterisk (*), percentages on BP-20. <sup>b</sup>Retention indices measured on BP-1 <sup>c</sup>Retention indices measured on BP20 capillary column. tr = trace level (<0.05%). MS: components identified by MS on apolar and/or polar column. NMR (plain) = identification in EO and/or ME and/or HE. NMR (italic) = identification in fractions of CC. <sup>d</sup>Percentages calculated by combination of GC and NMR.</footnote>
various sesquiterpenes previously identified by MS and overall permitted the identification, in total, of two dozen components, mainly sesquiterpenols not previously suggested by MS.

**Chemical composition of the EO:** Leaves of *F. japonica* produced, by HD, a yellow EO (yield: 0.39% v/w, on a dry weight basis). The EO was analysed by GC(RI), GC-MS and ¹³C NMR, then subjected to CC and the fractions were analyzed by GC(RI) and ¹³C NMR. In total, 60 components (91.6% of the EO) were identified (Table 1). The EO exhibited a high level of sesquiterpenes bearing various skeletons. The major component was germacrene D (14.9%) accompanied by various oxygenated sesquiterpenes bearing a tertiary alcohol function that accounted for almost half (47%) of the EO composition, for example, β-elemol (9.1%), *cis*-guai-6-en-10β-ol (6.3%), β-eudesmol (5.5%), α-eudesmol (4.4%), and eremophil-9-en-11-ol (4.2%). Sesquiterpene hydrocarbons were also represented by δ-elemene (5.2%), β-elemene (3.3%) and (E)-β-caryophyllene (1.6%). Limonene is the unique monoterpene identified in appreciable amount (7.1%). Some sesquiterpenols are uncommon components of *Fortunella* EO and more generally of Citrus and they contribute to the originality of *F. japonica* leaf oil: germacra-1(10),5-dien-4α-ol, amorph-4-en-10α-ol, amorph-4-en-10β-ol, and *trans*-guai-6-en-10β-ol. The composition of the investigated *F. japonica* leaf oil differed substantially from those of other origins: Vietnam (elemol, 17.7%; β-eudesmol, 16.6%; epi-bicyclosesquiphellandrene, 16.6%) [10]; Egypt [δ-cadinene (up to 16.0%), guaiol (up to 11.7%), elemol (up to 19.4%), γ-eudesmol (up to 11.9%), and τ-cadinol (up to 11.1%)] [11]. Obviously it differed drastically from the Nepalese leaf containing mostly oxygenated monoterpenes, linalool and geraniol [8] and the leaf oil from Thailand whose composition is dominated by β-pinene [9].

**Chemical composition of the ME:** Leaves of *F. japonica* from the same batch were submitted to MAHD and yielded a dark yellow extract (yield: 0.09%, v/w, on dry weight basis). The ME was treated like the EO. In total 52 components (93.8% of the ME) were identified (Table 1). The content of oxygenated sesquiterpenes, already important in the EO, appears to be higher in the ME (65.8% vs. 46.9%). Indeed, oxygenated compounds possess a high electric dipolar moment. They are also more easily extracted than hydrocarbons possessing a low dipolar moment. However, each sesquiterpene bearing a tertiary alcohol responded in a different manner to MAHD extraction. For instance, the content of some sesquiterpenols increased substantially in the ME compared with the EO: germacra-1(10),5-dien-4α-ol (7.1% vs. 2.4%), β-elemol 12.4% vs. 9.1%), *cis*-guai-6-en-10β-ol (9.0% vs. 6.3%), β-eudesmol (8.2% vs. 5.5%), and (E)-phytol (3.8% vs. 0.3%).

**Chemical composition of the HE:** During the hydrodistillation process, EO is constantly crossed by a water flux. This aqueous phase called hydrolate is permanently enriched with water soluble compounds. During HD, the hydrolate was collected and then extracted with Et₂O. The HE was analysed by GC(RI), GC-MS and ¹³C NMR. In total 28 components, accounting for 93.3% of the HE, were identified (Table 1). The chemical composition of this extract differed drastically from those of EO and ME. Indeed, cryptomeridiol (23.3%), absent from the EO and ME, became the main component of the HE. The content of other sesquiterpenols was strongly increased: β-eudesmol (20.6%) and α-eudesmol (10.7%). The content of sesquiterpenes bearing a tertiary alcohol function was again increased and reached 92.3%. In parallel, germacrene D, the major compound of the EO (14.9%) accounted for 0.1% of the HE. Solubility is the major phenomenon responsive for qualitative and quantitative variations between EO and HE

compositions. Cryptomeridiol could result from the hydration of eudesmol isomers in the slightly acidic conditions (pH = 5.5) of HD.

In conclusion, EO from *F. japonica* appears to be rich in oxygenated sesquiterpenes that bear a tremendous variety of skeletons. The use of a green extraction technique (MAHD) produced quickly an extract with higher content of oxygenated sesquiterpenes. The high level of oxygenated compounds is often associated with biological activities, which is an interesting key point of this extraction technique. However the low yield observed with MAHD in this study is a disadvantage compared with traditional HD. Hydrolate, a by-product of HD, displayed the highest content of oxygenated compounds and as the major component a sesquiterpene diol totally absent from the EO and ME. From an analytical point of view, combined analysis by chromatographic and spectroscopic techniques (including ¹³C NMR) appeared useful for identification of sesquiterpenes bearing a tertiary alcohol function and diverse skeletons.

## Experimental

**Plant material:** *F. japonica* trees were grown in the INRA-CIRAD citrus collection (certified as CRB citrus NF96-600, localized in San Ghjulianu, Corsica, France). Details previously reported in reference [2].

**Hydrodistillation (HD):** About 300 g of mature leaves from spring flush of *F. japonica* were dried one day at room temperature, away from direct sunlight and then they were subjected to water-distillation (3 h), using a Clevenger-type apparatus.

**Microwave assisted hydrodistillation (MAHD):** MADH was performed using the 'DryDist' microwave oven (multimode microwave reactor, 2.45 GHz, maximum delivered power of 1000 W, variable in 10 W increments). Temperature was monitored using an external infrared (IR) sensor. In a typical MADH procedure performed at atmospheric pressure, 300 g of mature leaves from spring flush of *F. japonica* were heated using a fixed power density of 1 W/g for 45 min with water as extraction solvent. A cooling system outside the microwave cavity condensed the distillate continuously. Condensed water was refluxed to the extraction vessel in order to provide uniform conditions of temperature and humidity for extraction. The following extraction program was used: 20 s at 400 W/200°C - 2 min at 600W/120°C and 30 min at 550 W/120°C.

**Hydrolate extraction:** Fractions of 20 mL of hydrolate were collected every 15 min during the HD process. The total amount of hydrolate (320 mL) was extracted using Et₂O (3 x 30 mL). The organic layer was dried over MgSO₄ and the solvent removed under reduced pressure to obtain the hydrolate extract (HE).

**Fractionation of EO and ME:** An aliquot (1.845 g) of EO was fractionated by CC (silica gel, 63-200 µm, 80 g) and 11 fractions (F1-F11) were eluted with a gradient of solvents of increasing polarity, *n*-pentane/Et₂O (P/Et₂O): F1-F6: P; 0.142, 0.135, 0.125, 0.126, 0.113, 0.055 g; F7-F11: P:Et₂O, 95:5 to 0:100; 0.280, 0.305, 0.250, 0.122, 0.117 g. An aliquot (2.055 g) of ME leaf extract was fractionated by CC (silica gel, 63-200 µm, 50 g) and 8 fractions (F'1-F'8) were eluted with a gradient of solvents of increasing polarity, *n*-pentane/diethyl ether (P/Et₂O): F'1:P, 0.683g; F'2-F'8 (P/Et₂O from 85/15 to 0/100): F'2-F'8: 0.137; 0.133; 0.265; 0.162; 0.170; 0.458; 0.048 g, respectively.

**GC (FID) and ¹³C NMR analysis:** Analyses were carried out as previously reported [15].
**GC-MS analysis:** The EO, ME and HE were analysed with a Perkin-Elmer TurboMass detector (quadrupole), directly coupled to a Perkin-Elmer Autosystem, equipped with two fused silica capillary columns (50 m x 0.22 mm x 0.25 µm film thickness), BP-1 (polydimethyl siloxane) and BP-20 (polyethylene glycol). Carrier gas, helium at 0.8 mL/min; split, 1/75; injection volume, 0.5 µL; injector temperature, 250°C; oven temperature programmed from 60°C to 220°C at 2°C/min and then held isothermal (20 min); Ion source temperature, 250°C; energy ionization, 70 eV; EI mass spectra were acquired over the mass range 40-400 Da.

**Identification of components:** Identification of the individual components was based: i) on comparison of their GC retention indices (RI) on apolar and polar columns with those of authentic compounds or with literature data ii) on computer matching with a laboratory-made and commercial mass spectral libraries and comparison of spectra with literature data [16], iii) on comparison of the signals in the ¹³C NMR spectra of the mixtures with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software [14].

**Acknowledgments** – We are indebted to the ‘Collectivité Territoriale de Corse’ for a research grant (SS).

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