# Chemical Variability and Anti-Inflammatory Activity of Rosmarinus officinalis L. Leaf Essential Oil from Algerian Sahara

Ahlem Bekhechi, Charaf Eddine Watheq Malti, Brahim Babali, Miloud Bouafia, Chahrazed Bekhechi, Joseph Casanova, Mathieu Paoli, Félix Tomi

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## To cite this version:

Ahlem Bekhechi, Charaf Eddine Watheq Malti, Brahim Babali, Miloud Bouafia, Chahrazed Bekhechi, et al.. Chemical Variability and Anti-Inflammatory Activity of Rosmarinus officinalis L. Leaf Essential Oil from Algerian Sahara. Chemistry and Biodiversity, 2024, 21 (4), 10.1002/cbdv.202302077. hal-05034550

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# Chemical Variability and Anti-inflammatory activity of *Rosmarinus officinalis* L. Leaf Essential Oil from Algerian Sahara

Ahlem Bekhechi,[a] Charaf Eddine Watheq Malti,[b, c] Brahim Babali,[d] Miloud Bouafia,[e] Chahrazed Bekhechi,[c] Joseph Casanova,[a] Mathieu Paoli,[a] and Félix Tomi*[a]

The chemical composition of 71 oil samples from the leaves of *Rosmarinus officinalis* L., harvested in three provinces: Naâma (Western Algeria), Béchar and Adrar (Algerian Sahara), was investigated by GC-FID, GC/MS and ¹³CNMR. In total, 52 compounds were identified accounting for 88.8% to 99.9% of the total composition. The chemical composition of the oils was largely dominated by monoterpenes, with 1,8-cineole (9.7-70.2%), camphor (0.3-31.0%) being the major compounds followed by borneol (0.3-21.0%), α-pinene (4.5-14.5%), β-pinene (0.1-12.0%), linalool (0.7-9.9%) and verbenone (up to

11.1%) which was present only in the samples harvested in Adrar. All compositions (71 samples) were submitted to statistical analysis. Combination of hierarchical clustering denrogram and principal component analysis suggested the existence of three groups (one of these being subdivided into two sub-groups) which were distinguished on the basis of 1,8-cineole, camphor and verbenone contents. Four essential oil samples, containing 1,8-cineole and/or camphor as main components, exhibited anti-inflammatory activity against lip-oxygenase, with IC₅₀ values in the range 93 to 155 µg/mL.

## Introduction

The genus *Rosmarinus* belonging to the Lamiaceae family, is very fragrant perennial woody shrubs or sub-shrubs. In Algeria, two species are mentioned: *Rosmarinus officinalis* L. and an endemic species namely *R. tournefortii* de Noé (synonym, *R. eriocalyx*). *Rosmarinus officinalis*, commonly known in Algeria as "Klil" or "Lazir" has inflorescences and calyx with very short pruinose hairs, constituted by tightly applied hairs. Inflorescences are in very short spikes, with squamiform bracts 1-2 mm,

quickly caduceus.[1] *R. officinalis*, an evergreen shrub usually growing in the garrigue and thin forest, is widespread throughout the Mediterranean basin and widely cultivated; it extends from Portugal to Turkey in the Northern side and from Eastern Morocco to Cyrenaica (Libya) in the Southern side. It is found in various bioclimatic stages, from sub-humid to the upper arid.[2] As an aromatic species, rosemary is widely used in the Mediterranean culinary as spice and as food flavoring. In Algeria (Naâma, Béchar and Adrar provinces), *R. officinalis* has been used in folk medicine for many purposes such as stomachache, menstrual problems, antihypertensive, cholagogue. It is also advised to treat cough, influenza, cold, rheumatism, muscle spasm, cholelithiasis, fever and memory.[3,4]

Widely used in aromatherapy, rosemary essential oil is the first "chemotyped" essential oil. According to the literature, it is classified in three chemotypes, namely, cineoliferum (high content in 1,8-cineole), camphoriferum (high content of camphor)[5] and verbenoniferum (fair content of verbenone) from Corsica and Sardinia.[6] *R. officinalis* chemotypes display specific biological activities: ct. cineole is used against pulmonary affections, bronchitis, sinusitis, and asthma, ct. camphor is used as antalgic, muscular relaxant, and against rheumatism pains, and lastly ct. verbenone purify blood, drains toxins, and it is appreciated as lypolitic.

The chemical composition of essential oil samples isolated from Algerian *R. officinalis* has been investigated. In general, aerial parts of *R. officinalis* were harvested at full flowering. However, most papers reported the chemical composition of a unique oil sample, mostly harvested on the wild and scarcely cultivated.[7-22] A few papers concerned 2-6 oil samples.[23,24-30] Lastly, a study investigated the composition of 15 oil samples, however no statistical analysis was performed.[31] Otherwise, various studies concerned the composition as a function of the

<footnote>[a] A. Bekhechi, J. Casanova, M. Paoli, F. Tomi
Université de Corse-CNRS, UMR 6134 SPE, Route des Sanguinaires, 20000 Ajaccio, France
E-mail: tomi_f@univ-corse.fr</footnote>

<footnote>[b] C. E. W. Malti
Faculté des Sciences et Technologies, Université Ahmed Zabana, Cité Bourmadia, 48000 Relizane, Algérie</footnote>

<footnote>[c] C. E. W. Malti, C. Bekhechi
Laboratoire de Produits Naturels, Département de Biologie, Université Abou Bekr Belkaid, Imama, 13000 Tlemcen, Algérie</footnote>

<footnote>[d] B. Babali
Laboratoire d'Ecologie et Gestion des Ecosystèmes Naturels, Département d'Ecologie et Environnement, Université Abou Bekr Belkaid, Imama, 13000 Tlemcen, Algérie</footnote>

<footnote>[e] M. Bouafia
Laboratoire Antibiotiques, Antifongiques, Physico-chimique, Synthèse et Activité Biologique, Département de Biologie, Université Abou Bekr Belkaid, Imama, 13000 Tlemcen, Algérie</footnote>

<footnote>Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cbdv.202302077</footnote>

<footnote>© 2024 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
plant life cycle,[32] and the isolation of rosemary oils by different processes.[33–36]

All these studies demonstrated a fair chemical variability of Algerian *R. officinalis* essential oil, although the occurrence of chemotypes was not evidenced by statistical analysis. In short, essential oil samples could be divided into two main groups. The first group contains all the essential oil samples whose composition is strongly dominated by a major component (with a content at least twice that of the second component) i) 1,8-cineole (30–73%),[7–9,20,21,23,26,28–31,33,34] ii) α-pinene (28.5–51.8%),[27,31,35] iii) camphor (33.1–52.1%).[23,26,31,36] The remaining oil samples displayed two-three components with more or less similar contents i) α-pinene/cineole/camphor,[7,11,12,14,18,24,26,33] ii) α-pinene/camphene/camphor,[19,26,27,30,31] iii) α-pinene/camphor,[24,27] iv) cineole/camphor,[10,22,25,33] v) miscellaneous.[15] A few oil samples contained verbenone at fair content[11–13,16,23,24,27,35] while (E)-β-caryophyllene was the unique sesquiterpene present at appreciable content.[10,17,23]

It could be pointed out that all the investigated samples were isolated from plants harvested in the North part of Algeria, from Tlemcen and Oran, then Algiers and the North-Central Algeria, to Annaba and the Tunisian frontier. Lahgouat, El Oued and El Bayadh were the localities of harvest located most southerly, in the North part of Sahara (approximately at 300 km South of Algiers).

In continuation of our on-going work devoted to the valorization of aromatic and medicinal plants from Algeria,[37]

the aim of the present study was to investigate the chemical composition of essential oils isolated from *R. officinalis* growing wild or cultivated in the Algerian Sahara, namely in Naâma, Béchar and Adrar provinces and to evidence an homogeneity or a chemical variability of the compositions. In addition, the anti-inflammatory activity of four samples, chosen according to their chemical profiles, was assessed by *in vitro* lipoxygenase inhibition assay.

## Results and Discussion

Aerial parts of 71 individual plants from *R. officinalis* have been harvested in three provinces of Algerian Sahara: Naâma (Saharan Atlas, arid area, 26 samples, N1–N26, wild plants), Béchar (Great Western Erg, arid area, 35 samples, B1-B35, wild plants) and Adrar (Hyper-arid area, 10 samples, cultivated plants, A1–A10) (Figure 1). Leaves of individual plants have been hydrodistilled using a Clevenger-type apparatus, yielding colorless essential oil samples.

### Essential oil yield

The essential oil yields (w/w) calculated from dry material varied drastically from sample to sample ranging from 0.66 to 2.76% (Table 1). The highest yields were obtained for Béchar and

<figure><img src="image_5.png" /><figcaption>Figure 1. Sampling provinces of *R. officinalis* from Algeria.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Table 1. Yields (%) of essential oils isolated from leaves of *R. officinalis* harvested in three provinces of Algerian Sahara.</figcaption></figure>

<table><thead><tr><th></th><th>Mean</th><th>SD<sup>a</sup></th><th>Min<sup>b</sup></th><th>Max<sup>c</sup></th><th>Mean</th><th>SD<sup>a</sup></th><th>Min<sup>b</sup></th><th>Max<sup>c</sup></th></tr></thead><tbody><tr><td>Naâma</td><td>May (10 samples)</td><td></td><td></td><td></td><td>November (16 samples)</td><td></td><td></td><td></td></tr><tr><td></td><td>2.09</td><td>0.50</td><td>1.30</td><td>2.76</td><td>1.71</td><td>0.56</td><td>0.85</td><td>2.49</td></tr><tr><td>Béchar</td><td>April (21 samples)</td><td></td><td></td><td></td><td>November (14 samples)</td><td></td><td></td><td></td></tr><tr><td></td><td>2.00</td><td>0.38</td><td>1.11</td><td>2.49</td><td>1.67</td><td>0.36</td><td>0.77</td><td>2.18</td></tr><tr><td>Adrar</td><td>July (4 samples)</td><td></td><td></td><td></td><td>October (6 samples)</td><td></td><td></td><td></td></tr><tr><td></td><td>0.83</td><td>0.09</td><td>0.76</td><td>0.95</td><td>0.70</td><td>0.04</td><td>0.66</td><td>0.74</td></tr></tbody></table>

<footnote><sup>a</sup>: Standard deviation (SD); <sup>b</sup>: Minimum (Min), <sup>c</sup>: Maximum (Max).</footnote>
Naàma provinces (0.77–2.76%) and the lowest were observed for cultivated plants harvested in Adrar (0.66–0.95%). However, an appreciable variation was observed between the two harvest periods. Indeed, the yield values obtained in spring were more or less 15–20% higher than those observed in autumn.

## Chemical analysis of four selected essential oil samples

All the essential oil samples were submitted to GC analysis in combination with retention indices (RIs) on two chromatographic columns of different polarity; 16 and 10 samples, chosen according to their chemical profiles, were analyzed respectively by GC/MS and by ¹³C NMR following a computerized method developed at the University of Corsica.[³⁸⁻⁴⁰] The detailed compositions of four oil samples (two cineole-rich oil samples and two camphor-rich oil samples) are reported in Table 2, including also the mode of identification of every component. In total, 52 components have been identified. They accounted for 91.4–98.7% of the whole composition. As expected, all the samples contained mainly oxygenated monoterpenes as well as monoterpene hydrocarbons. Sesquiterpene hydrocarbons and oxygenated sesquiterpenes were scarce and always at low content.

Concerning oil samples B18 and B29, from Béchar, 1,8-cineole (60.1 and 53.1%) dominated strongly the chemical composition. Linalool (9.6 and 5.7%) and α-terpineol (4.8 and 4.6%) were present at fair content while camphor (1.1 and

11.6%) varied substantially from sample to sample. α-Pinene (5.8 and 7.1%) and β-pinene (4.2 and 3.7%) represented the monoterpene hydrocarbons. (E)-β-Caryophyllene and α-humulene as well as the corresponding oxides were found at very low content.

Concerning oil samples A2 and A3 from Adrar, camphor (23.2 and 22.9%) was the main component followed by 1,8-cineole (13.2 and 14.4%), borneol (10.5 and 8.8%), and verbenone (7.6 and 10.2%). Verbenone (7.6 and 10.2%), as well as campholenol (1.3 and 1.4%) were not detected in the oil samples from Béchar. α-Pinene (10.0 and 9.0%) was the unique monoterpene hydrocarbon present at appreciable content, accompanied by camphene (4.7 and 3.2%) and limonene (4.0 and 3.2%), the last two compounds, were present in low amount (0.8 and 1.3%; 1.0 and 1.5%, respectively) in the samples from Béchar. (E)-β-Caryophyllene and α-humulene as well as the corresponding oxides were found at trace level.

## Chemical variability of *R. officinalis* from Algerian Sahara

Data obtained from the chemical composition of the 71 samples have been submitted to statistical analysis, Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). Three groups were differentiated, they contained 10, 30 and 31 oil samples, respectively (Figure 2). Group III has been divided into two sub-groups, IIIA, (23 oil samples) and IIIB (8 oil samples).

<figure><img src="image_7.png" /><figcaption>**Figure 2.** Hierarchical Cluster Analysis of the compositions of the 71 essential oil samples isolated from leaves of *R. officinalis* from Algerian Sahara.</figcaption></figure>
<table><caption>Table 2. Chemical composition of four essential oil samples isolated from leaves of Saharan *R. officinalis*.</caption><thead><tr><th></th><th>Compounds<sup>a</sup></th><th>Rla-lit<sup>c</sup></th><th>Rlp-lit<sup>c</sup></th><th>Rla<sup>b</sup></th><th>Rlp<sup>b</sup></th><th>B18</th><th>B29</th><th>A2</th><th>A3</th><th>Identification</th></tr></thead><tbody><tr><td>1</td><td>Tricyclene</td><td>922</td><td>1012</td><td>921</td><td>nd</td><td>Tr</td><td>Tr</td><td>0.2</td><td>0.2</td><td>RI, MS</td></tr><tr><td>2</td><td>α-Thujene</td><td>926</td><td>1026</td><td>924</td><td>1020</td><td>0.1</td><td>0.1</td><td>-</td><td>-</td><td>RI, MS</td></tr><tr><td>3</td><td>α-Pinene</td><td>936</td><td>1026</td><td>932</td><td>1020</td><td>5.8</td><td>7.1</td><td>10.0</td><td>9.0</td><td>RI, MS <sup>13C NMR</sup></td></tr><tr><td>4</td><td>α-Fenchene</td><td>945</td><td>1061</td><td>943</td><td>1060</td><td>Tr</td><td>Tr</td><td>0.1</td><td>0.1</td><td>RI, MS</td></tr><tr><td>5</td><td>Camphene</td><td>947</td><td>1068</td><td>945</td><td>1071</td><td>0.8</td><td>1.3</td><td>4.7</td><td>3.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>6</td><td>Verbenene</td><td>946</td><td>1124</td><td>947</td><td>1131</td><td>-</td><td>-</td><td>0.6</td><td>0.5</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>7</td><td>1-Octen-3-ol</td><td>966</td><td>1444</td><td>964</td><td>1452</td><td>0.1</td><td>0.2</td><td>0.3</td><td>0.5</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>8</td><td>3-Octanone</td><td>966</td><td>1265</td><td>966</td><td>1259</td><td>-</td><td>-</td><td>Tr</td><td>0.1</td><td>RI, MS</td></tr><tr><td>9</td><td>Sabinene</td><td>973</td><td>1122</td><td>968</td><td>1126</td><td>0.1</td><td>0.1</td><td>-</td><td>-</td><td>RI, MS</td></tr><tr><td>10</td><td>β-Pinene</td><td>978</td><td>1110</td><td>972</td><td>1116</td><td>4.2</td><td>3.7</td><td>0.2</td><td>0.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>11</td><td>Myrcene</td><td>987</td><td>1161</td><td>981</td><td>1165</td><td>1.1</td><td>1.2</td><td>0.7</td><td>0.7</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>12</td><td>α-Phellandrene</td><td>999</td><td>1168</td><td>998</td><td>1170</td><td>0.1</td><td>0.1</td><td>Tr</td><td>0.1</td><td>RI, MS</td></tr><tr><td>13</td><td>δ-3-Carene</td><td>1007</td><td>1147</td><td>1006</td><td>1153</td><td>Tr</td><td>Tr</td><td>0.4</td><td>0.4</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>14</td><td>α-Terpinene</td><td>1011</td><td>1178</td><td>1010</td><td>1186</td><td>0.3</td><td>0.4</td><td>0.1</td><td>0.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>15</td><td>p-Cymene</td><td>1015</td><td>1270</td><td>1013</td><td>1277</td><td>0.7</td><td>0.8</td><td>2.3</td><td>1.6</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>16</td><td>Limonene*</td><td>1025</td><td>1198</td><td>1024</td><td>1206</td><td>1.0</td><td>1.5</td><td>4.0</td><td>3.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>17</td><td>1,8-Cineole*</td><td>1025</td><td>1211</td><td>1024</td><td>1216</td><td>60.1</td><td>53.1</td><td>13.2</td><td>14.4</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>18</td><td>cis-β-Ocimene</td><td>1029</td><td>1234</td><td>1026</td><td>1237</td><td>0.4</td><td>0.1</td><td>-</td><td>-</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>19</td><td>γ-Terpinene</td><td>1051</td><td>1245</td><td>1049</td><td>1250</td><td>0.5</td><td>0.6</td><td>Tr</td><td>0.1</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>20</td><td>Sabinene hydrate<sup>f</sup></td><td>1056</td><td>1460</td><td>1056</td><td>1464</td><td>0.3</td><td>0.2</td><td>-</td><td>-</td><td>RI, MS</td></tr><tr><td>21</td><td>cis-Linalool oxide THF</td><td>1065</td><td>1446</td><td>1061</td><td>1443</td><td>Tr</td><td>Tr</td><td>0.1</td><td>0.1</td><td>RI, MS</td></tr><tr><td>22</td><td>Terpinolene</td><td>1079</td><td>1282</td><td>1079</td><td>1288</td><td>0.1</td><td>0.2</td><td>0.1</td><td>0.2</td><td>RI, MS</td></tr><tr><td>23</td><td>Linalool</td><td>1086</td><td>1544</td><td>1085</td><td>1550</td><td>9.6</td><td>5.7</td><td>3.8</td><td>4.8</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>24</td><td>Fenchol</td><td>1101</td><td>1570</td><td>1102</td><td>1584</td><td>-</td><td>-</td><td>0.2</td><td>0.7</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>25</td><td>Camphor</td><td>1125</td><td>1515</td><td>1123</td><td>1524</td><td>1.1</td><td>11.6</td><td>23.2</td><td>22.9</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>26</td><td>trans-Pinocarveol</td><td>1126</td><td>1661</td><td>1126</td><td>1659</td><td>-</td><td>-</td><td>0.3</td><td>0.2</td><td>RI, MS</td></tr><tr><td>27</td><td>trans-Sabinol</td><td>1131</td><td>1717</td><td>1126</td><td>1702</td><td>0.1</td><td>0.1</td><td>-</td><td>-</td><td>RI, MS</td></tr><tr><td>28</td><td>trans-Verbenol</td><td>1134</td><td>1680</td><td>1132</td><td>1682</td><td>0.1</td><td>0.1</td><td>0.3</td><td>0.3</td><td>RI, MS</td></tr><tr><td>29</td><td>Camphene hydrate</td><td>1136</td><td>1602</td><td>1135</td><td>1600</td><td>Tr</td><td>Tr</td><td>0.3</td><td>0.2</td><td>RI, MS</td></tr><tr><td>30</td><td>trans-Pinocamphone*</td><td>1144</td><td>1523</td><td>1141</td><td>1514</td><td>-</td><td>-</td><td>0.2</td><td>0.6</td><td>RI, MS</td></tr><tr><td>31</td><td>Pinocarvone*</td><td>1140</td><td>1575</td><td>1141</td><td>1574</td><td>Tr</td><td>0.1</td><td>0.4</td><td>0.4</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>32</td><td>δ-Terpineol</td><td>1148</td><td>1679</td><td>1149</td><td>1676</td><td>0.9</td><td>0.8</td><td>-</td><td>-</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>33</td><td>iso-Pinocamphone<sup>g</sup></td><td>1153</td><td>1536</td><td>1150</td><td>1550</td><td>0.1</td><td>-</td><td>1.3</td><td>1.6</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>34</td><td>Borneol*</td><td>1153</td><td>1700</td><td>1150</td><td>1705</td><td>2.0</td><td>2.2</td><td>10.5</td><td>8.8</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>35</td><td>p-Cymen-8-ol</td><td>1165</td><td>1848</td><td>1162</td><td>1850</td><td>-</td><td>Tr</td><td>0.1</td><td>0.3</td><td>RI, MS</td></tr><tr><td>36</td><td>Terpinen-4-ol</td><td>1164</td><td>1601</td><td>1163</td><td>1606</td><td>1.0</td><td>1.0</td><td>0.3</td><td>1.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>37</td><td>α-Terpineol</td><td>1176</td><td>1694</td><td>1174</td><td>1700</td><td>4.8</td><td>4.6</td><td>3.0</td><td>2.7</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>38</td><td>Verbenone</td><td>1184</td><td>1720</td><td>1184</td><td>1713</td><td>-</td><td>-</td><td>7.6</td><td>10.2</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>39</td><td>α-Campholenol<sup>e</sup></td><td>1185</td><td>1790</td><td>1187</td><td>1796</td><td>-</td><td>-</td><td>1.3</td><td>1.4</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>40</td><td>Citronellol</td><td>1212</td><td>1764</td><td>1211</td><td>1768</td><td>0.1</td><td>0.1</td><td>Tr</td><td>Tr</td><td>RI, MS</td></tr><tr><td>41</td><td>Carvone</td><td>1218</td><td>1734</td><td>1218</td><td>1739</td><td>-</td><td>-</td><td>0.2</td><td>0.2</td><td>RI, MS</td></tr><tr><td>42</td><td>Geraniol</td><td>1239</td><td>1839</td><td>1236</td><td>1838</td><td>Tr</td><td>Tr</td><td>0.2</td><td>0.2</td><td>RI, MS</td></tr><tr><td>43</td><td>Thymol</td><td>1272</td><td>2164</td><td>1261</td><td>2170</td><td>Tr</td><td>Tr</td><td>0.1</td><td>0.1</td><td>RI, MS</td></tr><tr><td>44</td><td>Bornyl acetate</td><td>1270</td><td>1580</td><td>1270</td><td>1584</td><td>0.4</td><td>0.3</td><td>0.5</td><td>0.7</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>45</td><td>Carvacrol</td><td>1283</td><td>2211</td><td>1278</td><td>2215</td><td>Tr</td><td>Tr</td><td>0.1</td><td>Tr</td><td>RI, MS</td></tr><tr><td>46</td><td>Piperitenone</td><td>1317</td><td>1909</td><td>1311</td><td>1926</td><td>-</td><td>-</td><td>0.4</td><td>0.4</td><td>RI, MS, <sup>13C NMR</sup></td></tr><tr><td>47</td><td>Methyleugenol</td><td>1376</td><td>2006</td><td>1370</td><td>2017</td><td>Tr</td><td>0.1</td><td>0.1</td><td>0.2</td><td>RI, MS</td></tr><tr><td>48</td><td>(E)-β-Caryophyllene</td><td>1419</td><td>1598</td><td>1417</td><td>1600</td><td>0.9</td><td>0.5</td><td>Tr</td><td>0.1</td><td>RI, MS, <sup>13C NMR</sup></td></tr></tbody></table>
<table><caption>**Table 2.** continued</caption><thead><tr><th></th><th>Compounds<sup>a</sup></th><th>Rla-lit<sup>c</sup></th><th>Rlp-lit<sup>c</sup></th><th>Rla<sup>b</sup></th><th>Rlp<sup>b</sup></th><th>B18</th><th>B29</th><th>A2</th><th>A3</th><th>Identification</th></tr></thead><tbody><tr><td>49</td><td>α-Humulene</td><td>1449</td><td>1668</td><td>1450</td><td>1671</td><td>0.4</td><td>0.3</td><td>Tr</td><td>Tr</td><td>RI, MS, <sup>13</sup>C NMR</td></tr><tr><td>50</td><td>δ-Cadinene</td><td>1506</td><td>1763</td><td>1515</td><td>1758</td><td>-</td><td>-</td><td>Tr</td><td>Tr</td><td>RI, MS</td></tr><tr><td>51</td><td>Caryophyllene oxide</td><td>1570</td><td>1986</td><td>1570</td><td>1983</td><td>0.3</td><td>0.3</td><td>Tr</td><td>Tr</td><td>RI, MS</td></tr><tr><td>52</td><td>Humulene oxide</td><td>1601</td><td>2020</td><td>1596</td><td>2039</td><td>0.1</td><td>0.2</td><td>-</td><td>Tr</td><td>RI, MS</td></tr><tr><td colspan="11"><hr></td></tr><tr><td colspan="11">Monoterpene hydrocarbons</td></tr><tr><td colspan="11">Oxygenated monoterpenes</td></tr><tr><td colspan="11">Sesquiterpene hydrocarbons</td></tr><tr><td colspan="11">Oxygenated sesquiterpenes</td></tr><tr><td colspan="11">Others</td></tr><tr><td colspan="11">Total (%)</td></tr><tr><td colspan="11">97.6 98.7 91.4 93.0</td></tr></tbody></table>

Mean value (M), standard deviation (SD) as well as minimum and maximum content have been reported in Table 3 for every component present at appreciable content. It could be highlighted that the index of dissimilarity was high and therefore, qualitative and quantitative differences between groups may be discussed.

The main group (Group II) is constituted of 30 essential oil samples whose composition is strongly dominated by 1,8-cineole (M = 62.3%, SD = 4.04). Other components at appreciable content are (Mean, SD): α-pinene (5.9%, 0.95), β-pinene (4.9%, 1.15), camphor (3.4%, 2.0) and α-terpineol (4.4%, 0.53). Linalool reached highest values in this group (5.7%, 1.87).

In parallel, group III (31 oil samples) is similar in importance to the previous group. However, it could be divided in sub-groups of inequal importance, IIIA (23 samples) and IIIB (8 samples). In both sub-groups, cineole remained the main component, much more important in sub-group IIIA (M = 47.9%, SD = 3.40) than in sub-group IIIB (M = 37.6%, SD = 7.23). In contrast, camphor is more abundant in sub-group IIIB (M = 22.1%, SD = 4.31) than in sub-group IIIA (M = 9.1%, SD = 3.66). Other components present at appreciable content in both sub-groups were α-pinene, β-pinene and borneol.

Lastly, group I that contained 10 oil samples displayed camphor as main component (M = 21.2%, SD = 1.81) beside α-pinene (M = 10.5%, SD = 1.76), cineole (M = 13.6%, SD = 1.46), borneol (M = 9.0%, SD = 0.84), and overall, verbenone (M = 10.0%, SD = 0.98), which was totally missing in all the other samples.

Concerning the repartition of essential oil samples with respect to their geographical area, it could be highlighted, going to north to south, that:

i) Most oil samples (19 out of 26) isolated from plant harvested in Naâma region belong to sub-group IIIA, characterized by a high content of 1,8-cineole (M = 47.9%, SD = 3.40), five oil samples belong to sub-group IIIB and the last two are contained in Group II;

ii) Most oil samples (28 out of 35) isolated from plant harvested in Béchar region belong to sub-group II, very rich in 1,8-cineole ((M = 62.3%, SD = 4.04), the other samples being distributed in sub-groups IIIA and IIIB;

iii) Lastly, the ten oil samples from Adrar region belonged to Group I, whose composition is dominated by camphor (M = 21.2%, SD = 1.81) beside cineole (M = 13.6%, SD = 1.46), α-pinene (M = 10.5%, SD = 1.76), verbenone (M = 10.0%, SD = 0.98), and borneol (M = 9.0%, SD = 0.84). It could be pointed out that verbenone was absolutely absent in the oil samples from Naâma and Béchar.

Going from Naâma, Béchar, and Adrar, it could be highlighted that oil samples from Naâma and Béchar belonged mainly to the cineole chemotype, while oil samples from the locality of harvest located most southerly, Adrar, all belonged to the camphor chemotype, although they contained appreciable amounts of verbenone, compound that characterize the third chemotype of *R. officinalis* essential oil. It should be kept in mind that the Adrar oil samples were isolated from cultivated plants of undetermined origin. Therefore, genetic factors may be responsible of the specific composition of the essential oil samples.

For comparison with previous works relative to Algerian *R. officinalis*, cineole-rich essential oils have been reported from plants harvested in Bordj Bou Arreridj (South-East Algiers),<sup>[7,26]</sup> Béjaïa and Tébessa (Eastern Algeria),<sup>[26,28]</sup> El-Bayadh,<sup>[21,29]</sup> Khenchela.<sup>[30]</sup> Camphor-rich EOs have been isolated from plants collected in Batna and M'Sila,<sup>[26]</sup> Constantine (North-East Algeria),<sup>[36]</sup> Djelfa and Laghouat.<sup>[23]</sup> *R. officinalis* oil samples containing fair amount of verbenone have been reported from plants harvested near Algiers, wild or cultivated,<sup>[23,27,35]</sup> Annaba, (Wild)<sup>[11]</sup> Sétif, (cultivated)<sup>[12]</sup> (Eastern Algeria), Tlemcen, (cultivated),<sup>[24]</sup> and overall, Mascara, (Western Algeria).<sup>[13]</sup> It could be pointed out that verbenone-containing essential oils from

<footnote><sup>a</sup>: Components have been listed following their order of elution on apolar column (BP-1); Percentages on apolar column, except those with an asterisk, % on polar column (BP-20); <sup>b</sup>: Rla, Rlp: Retention indices on apolar and polar columns, respectively; <sup>c</sup>: NMR: compound identified by <sup>13</sup>C NMR, at least in one oil sample; nd = not determined; Tr: traces (lower than 0.05%); <sup>d</sup>: Rla lit, Rlp lit, literature retention indices from Babushok et al.,<sup>[41]</sup> otherwise stated: <sup>e</sup>: ref [42]; <sup>f</sup>: ref [43]; <sup>g</sup>: cis stereochemistry of isopropyl and hydroxy groups.</footnote>
Algeria differed from those of Corsica and Sardinia by a lower content of bornyl acetate.[5]

### In vitro Anti-Inflammatory Activity

Inflammation is a physiological response to a variety of agents including infectious microorganisms, toxic chemical compounds and physical injury. There are many diseases that are associated with the inflammation process. So, chronic inflammation increases the risk for skin inflammation, autoimmune diseases such as arthritis and diabetes, Alzheimer's disease and various cancers. Lipoxygenases, nonheme iron-containing dioxygenases, catalyze the deoxygenating of polyunsaturated fatty acids to give unsaturated fatty acid hydroperoxides and help in the conversion of arachidonic acid into pro-inflammatory mediators called leukotrienes which are mediators of allergic response and inflammation.[44-46] Therefore, the elimination of inflammation may represent a valid strategy for cancer prevention and therapy. For that purpose, we evaluated the *in vitro* anti-inflammatory potential of four samples of *R. officinalis* leaf essential oil (B5, B34, N14 and A1) by determining their ability to inhibit lipoxygenase (LOX). The inhibition percentages of LOX are presented in Table 4.

<table><caption>**Table 4.** *In vitro* anti-inflammatory activity of *R. officinalis* leaf essential oil (Percentage Inhibition of LOX and IC₅₀ values).</caption><thead><tr><th>Samples</th><th>Concentrations (µl/mL)</th><th>Percentage Inhibition of LOX</th><th>IC<sub>50</sub> (µg/mL)</th></tr></thead><tbody><tr><td rowspan="4">A1</td><td>1.6</td><td>96.0 ± 0.7</td><td rowspan="4">93 ± 4.7</td></tr><tr><td>0.8</td><td>78.5 ± 0.8</td></tr><tr><td>0.4</td><td>68.1 ± 3.5</td></tr><tr><td>0.2</td><td>44.9 ± 1.2</td></tr><tr><td rowspan="3">B5</td><td>0.3</td><td>77.1 ± 4.6</td><td rowspan="3">115 ± 2.5</td></tr><tr><td>0.15</td><td>37.8 ± 2.6</td></tr><tr><td>0.075</td><td>27.1 ± 4.7</td></tr><tr><td rowspan="3">N14</td><td>0.4</td><td>84.2 ± 3.2</td><td rowspan="3">120 ± 3.9</td></tr><tr><td>0.2</td><td>72.2 ± 0.3</td></tr><tr><td>0.1</td><td>48.9 ± 3.6</td></tr><tr><td rowspan="4">B35</td><td>0.3</td><td>71.9 ± 1.9</td><td rowspan="4">155 ± 4.5</td></tr><tr><td>0.2</td><td>64.9 ± 7.8</td></tr><tr><td>0.1</td><td>33.6 ± 0.5</td></tr><tr><td>0.05</td><td>25.9 ± 0.5</td></tr><tr><td colspan="2">Concentrations (µg/mL)</td><td>Percentage Inhibition of LOX</td><td>IC<sub>50</sub> (µg/mL)</td></tr><tr><td rowspan="5">NDGA</td><td>50</td><td>89.8 ± 0.5</td><td rowspan="5">11 ± 0.5</td></tr><tr><td>25</td><td>74.6 ± 1.3</td></tr><tr><td>12.5</td><td>53.6 ± 1.8</td></tr><tr><td>6.25</td><td>36.3 ± 1.3</td></tr><tr><td>3.125</td><td>13.2 ± 0.7</td></tr></tbody><tfoot><tr><td colspan="4">NDGA: Nordihydroguaiaretic acid.</td></tr></tfoot></table>

All essential oil samples exhibited an inhibition of LOX activity and have dose dependent, with good percentages of inhibition ranging between 25% and 96% (Table 4). Indeed, at 0.3 and 0.4 µL/mL, the four oil samples showed an appreciable percentage of inhibition in the range 68.1 to 84.2%, while this inhibition was very expressed for sample A1 (96.0% at 1.6 µL/mL). The IC₅₀ values (concentration at which 50% of the lipoxygenase was inhibited) were determined for the *R. officinalis* leaf essential oil and for the nordihydroguaiaretic acid (NDGA), which used as a reference in anti-inflammatory activity. The IC₅₀ values, presented in Table 4, allow us to evaluate and compare the effectiveness of essential oils. So, the strongest anti-inflammatory ability was noted for sample A1 (IC₅₀ = 93 µg/mL), which contained camphor as major compound (23.2%) accompanied by cineole (13.2%) and α-pinene (10.0%). This sample was also characterized by an appreciable amount of *p*-cymene (2.3% vs. 0.5-0.6%), limonene (4.0% vs. 0.8-1.9%) and iso pinocamphone (1.3% vs. 0.0%) which probably participated to this good anti-inflammatory activity of the sample A1. However, in comparison with standard anti-inflammatory (NDGA: IC₅₀ = 11 µg/mL), the essential oils of the samples tested were found to be less active.

The anti-inflammatory activity of *R. officinalis* essential oil has been investigated. For instance, Baylac and Racine[47] reported a moderate lipoxygenase inhibitory activity for essential oil of *R. officinalis* ct. 1,8-cineole, with IC₅₀ value ranging between 51 and 100 µg/mL, vs. a standard (NDGA, IC₅₀ value < 10 µg/mL).

In parallel, the anti-inflammatory activity of various cineole-rich essential oils has been investigated. Indeed, Viljoen et al,[48] evaluated the anti-inflammatory activity of essential oil of 17 indigenous *Agathosma* species. All the essential oils exhibited *in vitro* anti-inflammatory activity with that of *A. namaquensis* (cineole, 23.5%) being the most potent (IC₅₀ = 31.54 µg/mL) although it displayed a low activity by compared to the standard (NDGA: IC₅₀ value of 2.39 µg/mL). Similarly, Albano et al.[49] reported a poor activity for the cineole-rich (49 and 59%, respectively) essential oils of *T. mastichina* (IC₅₀ = 1084 µg/mL) and *Salvia officinalis* (IC₅₀ = 827.9 µg/mL), the IC₅₀ value of positive control (NDGA) was 63.7 µg/mL. On the other hand, *Foeniculum vulgare*, *Citrus sinensis*, *C. reticulata* and *Zanthoxylum* species essentials oils in which α-pinene, *p*-cymene, limonene, β-caryophyllene, undecan-2-one and tridecan-2-one predominated, showed strong lipoxygenase inhibitory effects.[47,49-50]

### Conclusions

Seventy-one essential oil samples have been isolated by hydro-distillation from *R. officinalis* plants growing wild or cultivated in the Algerian Sahara, the localities of harvest (particularly Béchar and Adrar) being located most southerly than any other reported in the literature. The compositions of 71 samples displayed a fair chemical variability, three groups (one group subdivided into two sub-groups) being differentiated on the basis of 1,8-cineole, camphor and verbenone contents. Four oil samples have been tested to investigate their anti-inflammatory
activity against lipoxygenase. IC₅₀ values ranged between 93 and 155 µg/mL compared with the standard NDGA, IC₅₀ value, 11 µg/mL. It could be highlighted that *R. officinalis* growing wild or cultivated in arid provinces produce secondary metabolites similar to those found in temperate countries. In the domain of EOs, this point looks encouraging in view of the climatic change.

# Experimental Part

## Plant Material

In total, leaves from seventy-one individual plants of *R. officinalis* were collected in three provinces of Algeria: Naâma, Béchar and Adrar (Figure 1). Twenty six wild plants were sampled at Naâma province (Mécheria: Djebel Antar; 33° 34' 54" N, 0° 17' 45" E) (in November 2021 and in May 2022: N1-N26); thirty five wild samples were collected at Béchar province (Beni Ounif: Djebel Antar, 33° 34' 53.0" N, 0° 17' 9.7" W) (in April 2019 and in November 2019: B1-B35) and ten cultivated plants were sampled at Adrar province (Adrar: Mraguen, 27° 56' 25.0" N, 0° 19' 27.8" W) (in July and October 2019: A1-A10). Identification of the plants was performed by Dr B. Babali (Laboratory of Ecology and Management of Natural Ecosystems, University of Tlemcen, Algeria). A voucher specimen has been deposited at the Laboratory of Natural Products (Department of Biology, University of Tlemcen) under the accession N° L. 2314.

## Oil Distillation

Dry leaves (30–300 g) were submitted to hydrodistillation for 2 h using a Clevenger-type apparatus. Yields have been calculated from dry material (w/w).

## Analytical GC

GC analyses were performed on a Perkin-Elmer Clarus 500 gas chromatograph (Flame Ionization Detector) equipped two fused silica capillary columns (50 mm×0.22 mm, 0.25 µm film thickness), BP-1 (polydimethyl siloxane) and BP-20 (polyethylene glycol). The oven temperature was programmed from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min, injector temperature: 250 °C; detector temperature: 250 °C; carrier gas: hydrogen (1.0 mL/min); split: 1/60. The relative proportions of the oil constituents were expressed as percentages obtained by peak area normalization, without using correcting factors. Retention indices (RI) were determined relative to the retention times of a series of n-alkanes with linear interpolation ("Target Compounds" software from Perkin-Elmer).

## GC/MS Analysis

The essential oils were analyzed with a Perkin-Elmer TurboMass detector (quadrupole), directly coupled to a Perkin-Elmer Autosystem XL, equipped with a fused-silica capillary column (50 mm×0.22 mm i.d., film thickness 0.25 µm), BP-1 (dimethyl poly-siloxane). Carrier gas, helium at 0.8 mL/min; split, 1/60; 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; electron ionization mass spectra were acquired over the mass range 40–400 Da.

## $^{13}$C NMR Analyses

$^{13}$C NMR analysis was performed on a Bruker AVANCE 400 Fourier Transform spectrometer operating at 100.623 MHz for $^{13}$C, equipped with a 5 mm probe, in deuterated chloroform (CDCl₃), with all shifts referred to internal tetramethylsilane (TMS). $^{13}$C NMR spectra were recorded with the following parameters: pulse width (PW), 4 µs (flip angle 45°); acquisition time, 2.73 s for 128 K data table with a spectral width (SW) of 220 000 Hz (220 ppm); CPD mode decoupling; digital resolution 0.183 Hz/pt. The number of accumulated scans ranged 2000–3000 for each sample (around 40 mg of oil in 0.5 mL of CDCl₃). Exponential line broadening multiplication (1.0 Hz) of the free induction decay was applied before Fourier transformation.

## Identification of Components

Identification of the components was based: (a) on comparison of their GC retention indices (RI) on polar and apolar columns, determined relative to the retention times of a series of n-alkanes with linear interpolation (Target Compounds software of Perkin-Elmer), with those of authentic compounds and with reference data[41] on computer matching against commercial mass spectral libraries[51–53] and (b) on comparison of the signals in the $^{13}$C NMR spectra of essential oils with those of reference spectra compiled in the laboratory spectral library, with the help of a laboratory-made software.[38–40] In the investigated samples individual components were identified by NMR at contents as low as 0.3–0.4%.

## Anti-inflammatory Activity

The anti-inflammatory capacity of four samples (One sample from each PCA-HCA group: B5, B34, N14 and A1) of *R. officinalis* essential oil was evaluated by *in vitro* lipoxygenase inhibition assay. Lipoxygenase from *Glycine max* (soybean) (EC 1.13.11.12) type I-B inhibiting activity was assayed spectrophotometrically as described by Bayala *et al*. [46] with slight modifications. It was performed by recording the rate of change in absorbance at 234 nm. Indeed, the increase of absorbance at 234 nm due to formation of 13-hydroperoxides of linoleic acid (substrate used for LOX inhibition activity assay).[46]

Briefly, 20 µL of the enzyme solution (at the final concentration of 200 U/mL) was prepared in boric acid buffer (0.2 M; pH 9.0), mixed with 20 µL of essential oil solution in DMSO (0.05 to 1.6 µL/mL, final concentration), and dissolved in 1940 µL of boric acid buffer and then incubated at 25 °C for 5 min. The reaction started by addition of 20 µL of substrate solution (linoleic acid, 25 mM) and the mixture was incubated for 4 min. Then placed in a boiling water bath and boiled for 3 min. The absorbance was measured at 234 nm. Nordihydroguaiaretic acid (NDGA) was used as reference standard. All tests were performed on triplicate. The percentage of lipoxygenase inhibition was calculated according to the following equation:

$$ \text{Inhibition \%} = \frac{\text{Absorbance control} - \text{Absorbance sample}}{\text{Absorbance control}} \times 100 $$

The IC₅₀ value was calculated by the concentration of *R. officinalis* EO inhibiting 50% of LOX activity.
## Data Analysis

Principal Components Analysis (PCA) and Hierarchical clustering (Ward's method) were performed by Xlstat (Adinsoft, France).[54]

## Supplementary Material

Chemical composition of the 71 oil samples.

## Author Contributions

Conceptualization: C.B., J.C. and F.T.; methodology: C.B. and F.T., sampling and extraction: M.B. and C.E.W.M.; plant identification: B.B.; investigation: formal analysis, A.B. and M.P.; original draft preparation: A.B., C.B., J.C. and F.T. writing review: C.B. and J.C.

## Acknowledgements

We acknowledge The French Embassy for fellowship awarded to A.B. We also thank all the farmers from locations for their kindness and help.

## Conflict of Interests

The authors declare no conflict of interest.

## Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Keywords:** Rosmarinus officinalis • essential oil composition • GC/MS • chemical variability • anti-inflammatory

[1] P. Quézel, F. Médail, in *Nouvelle flore de l'Algérie et des régions désertiques méridionales, Edition CNRS, Tome II, Paris, France*, 1963, pp.1169.

[2] IUCN, 'A guide to medicinal plants in North Africa', IUCN, 2005, 256.

[3] M. Bouafia, F. Amamou, M. Gherib, M. Benaissa, R. Azzi, S. Nemmiche, *Vegetos* 2021, 34, 654-662.

[4] B. Benarba, *J. Intercultural Ethnopharmacol.* 2016, 5, 320-330.

[5] J. Casanova, F. Tomi, ISTE Ltd. London, UK, 2018, 18.

[6] E. M. Napoli, G. Curcuruto, G. Ruberto, *Biochem. System. Ecol.* 2010, 38, 659-670.

[7] C. Boutekedjiret, F. Bentahar, R. Belabbes, J. M. Bessiere, *J. Essent. Oil Res.* 1998, 10, 680-682.

[8] O. Touafek, A. Nacer, A. Kabouche, Z. Kabouche, C. Bruneau, *Chem. Nat. Compd.* 2004, 40, 28-29.

[9] Z. Kabouche, N. Boutaghane, S. Laggoune, A. Kabouche, Z. Ait-kaki, K. Benlabed, *Int. J. Aromather.* 2005, 15, 129-33.

[10] S. Djeddi, N. Bouchenah, I. Settar, H. D. Skaltsa, *Chem. Nat. Compd.* 2007, 43, 487-90.

[11] R. Giordani, Y. Hadef, J. Kaloustian, *Fitoterapia* 2008, 79, 199-203.

[12] S. Zoubiri, A. Baaliouamer, *Food Chem.* 2011, 129, 179-182.

[13] H. Belkhodja, B. Meddah, A. Tir Touil, N. Şekeroğlu, P. Sonnet, *World J. Pharmacol.* 2016, 5, 108-119.

[14] S. Ksouri, S. Djebir, A. A. Bentorki, A. Gouri, Y. Hadef, A. Benakhla, *J. Mycol. Méd.* 2017, 27, 245-249.

[15] S. Mehalaine, O. Belfadel, T. Menasria, A. Messaili, *Phytothérapie* 2018, 16, S155-S163.

[16] R. Sarmoum, S. Haid, M. Biche, Z. Djazouli, B. Zebib, O. Merah, *Agronomy* 2019, 9, 214.

[17] N. Zouaoui, H. Chenchouni, A. Bouguerra, T. Massouras, M. Barkat, *NFS J.* 2020, 18, 19-28.

[18] D. Hezil, H. Benseghir, S. Benamrouche, S. Zaidi, R. Zineddine, A. Bessas, A. Sela, *Agricultura* 2021, 3, 119-120.

[19] S. Adouane, Y. Bouatrous, M. S. Mehaoua, J. Tudela, S. Mechaala, V. Tomas, *J. Crop Prot.* 2022, 11, 173-184.

[20] A. Benabdallah, S. Betina, S. Bouchentouf, M. Boumendjel, S. Bechkri, C. Bensouici, F. Nicoli, V. Marzia, C. Negro, L. De Bellis, S. Afr. *J. Bot.* 2022, 147, 501-510.

[21] H. A. Benchohra, M. M. Dif, A. Ayache, H. E. S. Medjaher, *Egypt. Acad. J. Biolog. Sci.* 2022, 14, 373-381.

[22] S. Bencharif-Betina, N. Benhamed, A. Benabdallah, H. Bendif, A. Benslama, C. Negro, G. Plavan, O. H. Abd-Elkader, L. De Bellis, *Separations* 2023, 10, 394.

[23] C. Boutekedjiret, B. Buatois, J. M. Bessiere, *J. Essent. Oil-Bear. Plants* 2005, 8, 65-70.

[24] F. Atik-Bekkara, L. Bousmaha, S. A. Taleb Bendiab, J. B. Boti, J. Casanova, *Biologie & Santé* 2007, 7, 6-11.

[25] N. Sahraoui, A. Hellal, C. Boutekedjiret, F. Bentahar, J. M. Bessière, *Int. J. Essent. Oil Ther.* 2007, 1, 83-90.

[26] T. Lograda, M. Ramdani, P. Chalard, G. Figueredo, *Glob. J. Res. Med. Plants Indig. Med.* 2013, 2, 794-807.

[27] T. Outaleb, M. Hazzit, Z. Ferhat, A. Baaliouamer, A. Yekkour, A. Zitouni, N. Sabaou, *J. Essent. Oil-Bear. Plants* 2015, 18, 654-665.

[28] L. Boutabia, S. Telailia, I. Bouguetof, F. Guenadil, A. Chefrour, *Bull. Soc. R. Sci. Liège* 2016, 85, 174-189.

[29] Z. Boukhobza, N. Boulenouar, A. Cheriti, Z. Kadri, *Nat. Vol. Essent. Oil* 2021, 8, 44-55.

[30] F. Z. Benomari, M. Sarazin, D. Chaib, A. Pichette, H. Boumghar, Y. Boumghar, N. Djabou, *Molecules* 2023, 28, 4439, 28114439.

[31] N. Hendel, E. Napoli, M. Sarri, A. Saija, M. Cristani, A. Nostro, G. Ginestra, G. Ruberto, *J. Essent. Oil-Bear. Plants* 2019, 22, 1-17.

[32] C. Boutekedjiret, R. Belabbes, F. Bentahar, J. M. Bessiere, *J. Essent. Oil Res.* 1999, 11, 238-240.

[33] C. Boutekedjiret, F. Bentahar, R. Belabbes, J. M. Bessiere, *Flavour Fragr. J.* 2003, 18, 481-84.

[34] C. Boutekedjiret, R. Belabbes, F. Bentahar, J. M. Bessière, S. A. Rezzoug, *J. Essent. Oil Res.* 2004, 16, 195-199.

[35] N. Bousbia, M. Abert Vian, M. A. Ferhat, E. Petitcolas, B. Y. Meklati, F. Chemat, *Food Chem.* 2009, 114, 355-362.

[36] A. Zermane, O. Larkeche, A.-H. Meniai, C. Crampon, E. Badens, C. R. Chim.* 2016, 19, 538-543.

[37] C. Bekhechi, C. E. W. Malti, M. Boussaid, I. Achouri, K. Belilet, M. Gibernau, J. Casanova, F. Tomi, *Nat. Prod. Commun.* 2019, 14, 1934578X19850030.

[38] F. Tomi, P. Bradesi, A. Bighelli, J. Casanova, *J. Magn. Reson. Anal.* 1995, 1, 25-34.

[39] Z. A. Ouattara, J. B. Boti, A. C. Ahibo, S. Sutour, J. Casanova, F. Tomi, A. Bighelli, *Flavour Fragr. J.* 2014, 29, 371-379.

[40] O. Bazzali, Tran Huy Thai, Tran Minh Hoi, Nguyen Sinh Khang, Nguyen Thi Hien, J. Casanova, A. Bighelli, F. Tomi, *Molecules* 2016, 21, 840-851.

[41] V. I. Babushok, P. J. Linstrom, I. G. Zenkevich, *J. Phys. Chem. Ref. Data* 2011, 40, 043101, 1-47.

[42] J. P. Mariotti, F. Tomi, J. Casanova, J. Costa, A. F. Bernardini, *Flavour Fragr. J.* 1997, 12, 147-151.

[43] M. Gonny, C. Cavaleiro, L. Salgueiro, J. Casanova, *Flavour Fragr. J.* 2006, 21, 99-106.

[44] G. Salud Pérez, S. Miguel Zavala, G. Lucina Arias, L. Miguel Ramos, *J. Essent. Oil Res.* 2011, 23, 38-44.

[45] S. M. A. Shah, M. Ashraf, I. Ahmad, S. Arshad, M. Yar, A. Latif *J. Med. Plant Res.* 2013, 7, 219-222.

[46] B. Bayala, I. H. N. Bassole, C. Gnoula, R. Nebie, A. Yonli, L. Morel, G. Figueredo, J.-B. Nikiema, J.-M. A. Lobaccaro, J. Simpore, *PLOS One* 2014, 9, e92122.

[47] S. Baylac, P. Racine, *Int. J. Aromather.* 2003, 13, 138-144.

[48] A. M. Viljoen, A. Moolla, S. F. van Vuuren, R. L. van Zyl, K. H. C. Başer, B. Demirci, T. Özek, T. H. Trinder-Smith, *J. Essent. Oil Res.* 2006, 18, 12-16.
[49] S. M. Albano, A. S. Lima, M. G. Miguel, L. G. Pedro, J. G. Barroso, A. C. Figueiredo, *Rec. Nat. Prod.* **2012**, *6*, 35–48.

[50] E. A. Tanoh, F. Nea, T. K. Kemene, M. Genva, M. Saive, F. Z. Tonzibo, M.-L. Fauconnier, *Molecules* **2019**, *24*, 2445.

[51] W. A. Konig, D. H. Hochmuth, D. Joulain, 'Terpenoids and Related Constituents of Essential Oils', Library of Mass Finder 2.1, Institute of Organic Chemistry, Hamburg, **2001**.

[52] National Institute of Standards and Technology, 'PC version of the mass spectral library', Norwalk, CT, USA, **2014**.

[53] R. P. Adams, 'Identification of Essential Oils Components by Gas Chromatography/Mass Spectroscopy', Carol Stream, Allured, 4th Edn., **2007**.

[54] P. Legendre, L. Legendre, *Numerical Ecology*, 2nd Edn., Elsevier Science, Amsterdam, **1998**.

Manuscript received: December 22, 2023

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