# Essential Oil of Citrus aurantium L. Leaves: Composition, Antioxidant Activity, Elastase and Collagenase Inhibition

Chahinez Oulebsir, Hakima Mefti-Korteby, Zahr-Eddine Djazouli, Bachar Zebib, Othmane Merah

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Chahinez Oulebsir, Hakima Mefti-Korteby, Zahr-Eddine Djazouli, Bachar Zebib, Othmane Merah. Essential Oil of Citrus aurantium L. Leaves: Composition, Antioxidant Activity, Elastase and Collagenase Inhibition. Agronomy, 2022, 12 (6), 10.3390/agronomy12061466. [https://doi.org/10.37032/aa.12061466](https://doi.org/10.37032/aa.12061466)

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# Essential Oil of *Citrus aurantium* L. Leaves: Composition, Antioxidant Activity, Elastase and Collagenase Inhibition

Chahinez Oulebsir<fnref n="1" />, Hakima Mefti-Korteby<fnref n="1" />, Zahr-Eddine Djazouli <fnref n="2" />,*, Bachar Zebib <fnref n="3" />
and Othmane Merah <fnref n="4" />,<fnref n="5" />,*

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**Citation:** Oulebsir, C.; Mefti-Korteby, H.; Djazouli, Z.-E.; Zebib, B.; Merah, O. *Essential Oil of *Citrus aurantium* L.*

Leaves: Composition, Antioxidant Activity, Elastase and Collagenase Inhibition. *Agronomy* **2022**, *12*, 1466.

https://doi.org/10.3390/agronomy12061466

Academic Editor: Antonios Chrysargyris

Received: 11 May 2022

Accepted: 15 June 2022

Published: 18 June 2022

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**Copyright:** © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Abstract:** Sour orange (*Citrus aurantium* L.), which belongs to the Rutaceae family, is used around the Mediterranean Sea for ornamental and agronomic purposes as a rootstock for the *Citrus* species. Peels and flowers, the most-used parts of *Citrus aurantium* L., have constituted a largely promising area of research for their many medicinal properties. However, the leaves of sour orange have not yet been studied extensively. The present study aimed at investigating the essential oil composition of sour orange leaves grown in Algeria and determining their antioxidant and anti-inflammatory properties. Essential oil composition of leaves harvested before flowering was determined by GC-MS. Total phenol content, antioxidant activities (DPPH) and elastase and collagenase inhibition were assessed. Forty-three volatile compounds were detected in essential oil from leaves with a yield of 0.57%. The major compounds were linalool, linalyl acetate and α-Terpineol. Results show that the total phenol content and antioxidant activity of essential oil are low, 3.48 ± 0.10 mg/g (Gallic Acid Equivalent/EO) and IC₅₀ > 10,000 mg·L⁻<fnref n="1" />, respectively. In contrast, EO present an interesting level of elastase and collagenase inhibition. This result emphasizes the potential interest of the essential oil of sour orange mainly in relation to its anti-aging mechanism.

**Keywords:** sour orange; anti-elastase; anti-collagenase; linalool; linalyl acetate; α-terpineol

## 1. Introduction

The growing interest in food safety is today gaining momentum for both consumers and the food industry as people are becoming more concerned about the multiple health and environmental impacts of foods. Natural sources of compounds used in cosmetics and pharmaceutics processing today play a prominent role; their multiple positive health and environmental impacts and benefits have made them the basic principles of what constitutes a healthy diet today and in the future [1,2].

Annual production of *Citrus* plants has reached more than 126 million tons [3], one fifth of which were produced in the Mediterranean region. Therefore, *Citrus* could be considered an economically important fruit tree crop. Appreciated for their fruits, *Citrus* species are rich in vitamins B9, C and E, antioxidants, essential oil (EO), coumarins and dietary fiber [3–6].

*Citrus* fruits are not only used as a dessert, but also for the preparation of functional foods and *Citrus* fruit-derived products such as jams and juices, as well as in the food industry around the world. The by-products of the industrial processing of products,

<footnote n="1">¹ Laboratoire de Recherche des Plantes Aromatiques et Médicinales, Département des Biotechnologies, Faculté des Sciences de la Nature et de la Vie, Université de Blida-1, Blida 09000, Algeria; lidiachahinez23@gmail.com (C.O.); hakimakorteby@hotmail.fr (H.M.-K.)</footnote>

<footnote n="2">² Department of Biotechnology, Faculty of Nature and Life Sciences, University of Blida 1, Blida 09000, Algeria</footnote>

<footnote n="3">³ Laboratoires Agronutrition SAS, 31390 Carbonne, France; b.zebib@agro-nutrition.fr</footnote>

<footnote n="4">⁴ Laboratoire de Chimie Agro-industrielle (LCA), Université de Toulouse, INRA (Institut National de la Recherche Agronomique), INPT, 31030 Toulouse, France</footnote>

<footnote n="5">5 Département Génie Biologique, Université Paul Sabatier, IUT A, 32000 Auch, France</footnote>

<footnote>* Correspondence: zahro2002@gmail.com (Z.-E.D.); othmane.merah@ensiacet.fr (O.M.)</footnote>
mainly peels, constitute an invaluable source for the production of EO. The latter are widely used in the cosmetic, fragrance, pharmaceutical and food industries [6,7].

The chemical composition of *Citrus* EO has been extensively studied and several compositional patterns owing to the species/cultivars, origin, climate, season, ripening stage, extraction and analytical methods have been published [8–10]. The chemical composition of EO of sour orange (*Citrus aurantium*) was assessed in different plant parts during different seasons. Many studies were focused on EO extract from *C. aurantium* peels and Limonene was found to be the major component [10–19]. In contrast, other studies carried out on EO extract from *C. aurantium* flowers showed that Linalool and Linalyl acetate are the main components [13,14,20,21]. However, the volatile oil constituents from leaves have not received much attention in the literature. Indeed, the few reported studies that focused on *C. aurantium* leaves [13,21,22] showed that linalool is the main component of EO. In addition, a large number of studies on *C. aurantium* were performed in Tunisia and Greece. To our knowledge, two studies were released in Algeria concerning *C. aurantium* peels and leaves and their antibacterial activities for foods applications [11] and antifungal properties [23]. Recently, Lin et al. [24] have explored the antioxidant and antibacterial activities of essential oils of several *Citrus* species. They have highlighted interesting antioxidant activities. Nevertheless, there are no reports concerning antioxidant and/or anti-elastase and anti-collagenase activities of EO of different organs of *C. aurantium*.

The main objective of this study is to investigate the EO composition of sour orange (*C. aurantium*) leaves from Algeria and to evaluate its antioxidant, anti-elastase and anticollagenase activities.

## 2. Materials and Methods

### 2.1. Plant Material

The samples of *Citrus aurantium* (L.) var *Amara* were collected during the phenological stage of the inflorescence buds swelling (51 of the BBCH scale) in March and April 2018, at the botanical garden of the Boufarik Regional Plant Protection Station, 36 km South of Algiers (36°34′00″ N and 2°55′00″ E) and at 63 m above sea level. Three samples were collected from three different trees within the same area (100 m²).

The harvested plant material was sealed in zipped plastic bags for transport to the laboratory of the Department of Biotechnology, Faculty of Natural and Life Sciences (University of Blida 1, Algeria). The samples were dried for 48 h at 30 °C in a ventilated oven. Grinding was performed with a Retsch knife mill (model SM100, Retsch, Eragny sur Oise, France). After grinding, the samples were used immediately for the extraction of essential oils.

### 2.2. Essential Oil Extraction and Yield Estimation

An aliquot of 250 g of the ground leaves (of each harvested sample) was used for the extraction of EO by hydrodistillation using a Clevenger type apparatus. Pursuant to the directives of the European Pharmacopoeia, the extraction was performed during three hours. The EO yield was calculated as follows:

$$ \text{Oil yield (\%)} = \frac{\text{Weight of essential oil (g)}}{\text{Weight of dry sample (g)}} \times 100 $$

The oil was dried using anhydrous sodium sulfate, stored in a sealed vial and brought to 4 °C pending analysis.

### 2.3. Chemical Characterization of EO by Coupling Gas Chromatography/Mass Spectrometry

Analysis of the volatile part was carried out by using gas-chromatography (GC-2014 Shimazu Gas Chromatograph, Courtaboeuf, France), equipped with a flame ionization detector AOC-20i autosampler (Kyoto, Japan). An RTX-5MS (5% diphenyl/95% dimethyl polysiloxane) GC column of 30 m × 0.25 mm × 0.25 µm from Restek (Palo Alto, CA, USA) was used in this study. The sample of EO was prepared with hexane at the ratio of 1:10 (v/v). The injector operates in split mode with a ratio of 1/50. The temperature of the
injector was maintained at 250 °C and that of the detector at 270 °C. As to the temperature of the column, the following sequence was performed: an initial temperature of 50 °C was maintained for one minute, then raised from 50 to 175 °C at a rate of 5 °C per minute. The temperature of 175 °C was maintained for 10 min, then raised from 175 to 250 °C in one step at a rate of 15 °C per minute [25].

Identification of the individual components was based on the comparison of retention indices (RI) calculated, on the polar and apolar columns, with those of authentic compounds or data from the literature (National Institute of Standards and Technology, 2008) and commercial libraries [26,27], as well as on the analysis of each mass spectrum of the constituent compounds, or were identified by comparing their retention indices with those already described in the literature [28–32].

## 2.4. Assessement of Total Phenolic Content

Total phenolic content was assessed with Folin-Ciocalteu reagent, according to Salachna et al. [33], and measured by spectrophotometry. One hundred microliters of EO extract were added to 0.2 mL of Folin-Ciocalteu reagent, 1 mL of sodium carbonate (at 20%) and 2 mL of distilled water. The samples were kept at 20°C in darkness for 1 h. The absorbance of samples was measured at 760 nm. The standard curve was performed on the basis of gallic acid and results were expressed as mg gallic acid equivalents (GAE) per g of EO.

## 2.5. DPPH Radical Scavenging Activity

DPPH (2,2-diphenyl-1-picrylhydrazyl, Carlo Erba, Val-de-Reuil, France) Radical Scavenging assay was used to evaluate antioxidant activity of sour orange EO according to Popovici et al. [34]. One hundred microliters of EO was mixed with 100 µL of a methanolic solution of DPPH at a concentration of 63.5 µM.

Following incubation for 30 min at 25 °C, absorbance was measured at 517 nm using a GQ-1300-UV-Vis spectrophotometer (UV—Vis-Cary 4000, Agilent, Les-Ulis, France). A blank test was also performed by applying the same procedure to a solution without the test solution and its absorbance was measured. The free radical scavenging activity of the EO was calculated as a percentage of inhibition pursuant to the following equation:

$$DPPH scavenging (%) = \frac{(Absorbance of control - Absorbance of sample)}{Absorbance of control} \times 100$$

The antioxidant activity of EO was determined by the calculation of the IC50 index, defined as the concentration of the test solution deemed necessary to reduce by 50% the initial concentration of DPPH. All measurements were made in triplicate.

## 2.6. ABTS [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)] Radical Scavenging Activity

The method used for the determination of ABTS•+ free radical scavenging activity was the approach presented by Baragan Ferrer et al. [35]. A 70 mM K₂S₂O₈ solution was also prepared. The radical cation ABTS•+ was obtained by adding 50 mL of ABTS stock solution to 200 µL of K₂S₂O₈ solution. The mixture was kept in the dark at room temperature for 16 h. For the evaluation of essential oils, the ABTS•+ solution was diluted with PBS to give an absorbance of 0.800 ± 0.030 at 734 nm. Three milliliters of ABTS•+ solution was mixed with 200 µL of essential oil of C. aurantium. The mixture was shaken vigorously and then stored in the dark at 30 °C for 6 min. A PBS solution was used as a blank sample. All determinations were performed in triplicate. The results were presented as milligrams of Trolox equivalents per gram (mg TE·g⁻¹). ABTS•+ free radical scavenging activity (Inhibition = I%) was finally calculated according to the following equation:

$$I\% = \frac{AB - AA}{AB} \times 100$$
where I is the inhibition of ABTS•+, in %; AB is the absorbance of the blank; AA is the absorbance of EO (after 10 min).

## 2.7. Determination of Collagenase and Elastase Inhibition

The collagenase activity (*Clostridium histolyticum*, Fisher, Illkirch, France) was carried out on an N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala substrate (VWR, Fontenay-sous-Bois, France) according to Wittenauer et al. [36] and Zemour et al. [37]. The reduction in absorbance was carried out at 335 nm for 20 min using a BioTek ELX800 microplate reader (Colmar, France). Collagenase activity was performed in triplicate. It is expressed as a percentage of inhibition relative to the control.

Porcine pancreatic elastase (Servilab, Le Mans, France) was used in this study on a substrate of N-*Succ*-Ala-Ala-Ala-p-nitroanilide (Servilab, Le Mans, France) [34,35]. The release of p-nitroaniline was measured at 410 nm with a BioTek ELX800 microplate reader (Colmar, France). Measurements were performed in triplicate and the activity was expressed as a percentage of inhibition relative to the control.

## 2.8. Statistical Analyses

For essential oil yield and composition, as well as for the measurement of all activities, mean values and standard deviations were calculated from at least three replicates using MS Excel 2003 (Microsoft-France, Issy-les Moulineaux, France). Statistical analysis was performed by using one-way analysis of the variance (ANOVA), followed by the Duncans' post hoc test to compare the means showing significant variation (*p* < 0.05).

# 3. Results and Discussion

## 3.1. Essential Oil Yield and Composition

Forty-three volatile compounds were detected in the EO of Algerian *C. aurantium* leaves whose major compounds, linalool, linalyl acetate and α-Terpineol, constitute more than 73% of the total components (Table 1). The chromatogram is provided in Figure S1.

<table><caption>**Table 1.** Essential oil composition of Algerian *Citrus aurantium* from leaves.</caption><thead><tr><th>Pics</th><th>T<sub>R</sub> (Min)</th><th>Constituants</th><th>%</th><th>RI<sub>literature</sub></th><th>RI<sub>exp</sub></th><th>Group</th></tr></thead><tbody><tr><td>1</td><td>5.4</td><td>2-Ethyl furan</td><td>0.01 ± 0.00</td><td>689</td><td>692</td><td>Fur</td></tr><tr><td>2</td><td>6.9</td><td>1-Hexanol</td><td>0.02 ± 0.00</td><td>799</td><td>803</td><td>Alc</td></tr><tr><td>3</td><td>7</td><td>α-Pinene</td><td>0.20 ± 0.01</td><td>925</td><td>923</td><td>M</td></tr><tr><td>4</td><td>7.8</td><td>α-Thylene</td><td>0.01 ± 0.00</td><td>926</td><td>925</td><td>M</td></tr><tr><td>5</td><td>8.9</td><td>Camphene</td><td>0.01 ± 0.00</td><td>943</td><td>945</td><td>M</td></tr><tr><td>6</td><td>9.2</td><td>β-Pinene</td><td>3.20 ± 0.02</td><td>945</td><td>947</td><td>M</td></tr><tr><td>7</td><td>10.1</td><td>Sabinene</td><td>0.40 ± 0.01</td><td>973</td><td>974</td><td>M</td></tr><tr><td>8</td><td>10.4</td><td>δ3-Χαρενε</td><td>0.01 ± 0.00</td><td>986</td><td>989</td><td>M</td></tr><tr><td>9</td><td>11.1</td><td>β-Myrcene</td><td>2.25 ± 0.01</td><td>989</td><td>992</td><td>M</td></tr><tr><td>10</td><td>11.8</td><td>α-Terpinene</td><td>0.03 ± 0.00</td><td>1008</td><td>1016</td><td>M</td></tr><tr><td>11</td><td>12.2</td><td>Limonene</td><td>0.71 ± 0.01</td><td>1023</td><td>1024</td><td>M</td></tr><tr><td>12</td><td>12.5</td><td>β-Phellandrene</td><td>0.05 ± 0.00</td><td>1023</td><td>1025</td><td>M</td></tr><tr><td>13</td><td>13</td><td>2-Hexanal</td><td>0.11 ± 0.01</td><td>1024</td><td>1028</td><td>A</td></tr><tr><td>14</td><td>13.7</td><td>Cis-β-Ocimene</td><td>0.81 ± 0.02</td><td>1027</td><td>1030</td><td>M</td></tr><tr><td>15</td><td>13.8</td><td>γ-Τερπινενε</td><td>0.05 ± 0.00</td><td>1028</td><td>1031</td><td>M</td></tr><tr><td>16</td><td>14.8</td><td>Trans-β-Ocimene</td><td>2.40 ± 0.02</td><td>1028</td><td>1032</td><td>M</td></tr><tr><td>17</td><td>15.4</td><td>p-Cymene</td><td>0.05 ± 0.00</td><td>1029</td><td>1033</td><td>M</td></tr><tr><td>18</td><td>18.3</td><td>Cis-Oxide linalool</td><td>0.10 ± 0.00</td><td>1059</td><td>1065</td><td>OM</td></tr><tr><td>19</td><td>23.5</td><td>Trans-Oxide linalool</td><td>0.05 ± 0.00</td><td>1072</td><td>1073</td><td>OM</td></tr><tr><td>20</td><td>25.1</td><td>Terpinolene</td><td>0.45 ± 0.01</td><td>1078</td><td>1079</td><td>M</td></tr><tr><td>21</td><td>29.8</td><td>Linalool</td><td>30.62 ± 0.04</td><td>1083</td><td>1084</td><td>OM</td></tr><tr><td>22</td><td>30.5</td><td>Terpinen-4-ol</td><td>0.15 ± 0.00</td><td>1124</td><td>1129</td><td>OM</td></tr><tr><td>23</td><td>32.9</td><td>α-Terpineol</td><td>9.57 ± 0.05</td><td>1173</td><td>1175</td><td>OM</td></tr><tr><td>24</td><td>33</td><td>Citronellol</td><td>0.05 ± 0.00</td><td>1212</td><td>1213</td><td>M</td></tr><tr><td>25</td><td>33.5</td><td>Nerol</td><td>2.01 ± 0.00</td><td>1214</td><td>1216</td><td>OM</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Pics</th><th>T<sub>R</sub> (Min)</th><th>Constituents</th><th>%</th><th>RI<sub>literature</sub></th><th>RI<sub>exp</sub></th><th>Group</th></tr></thead><tbody><tr><td>26</td><td>36.5</td><td>Neral</td><td>0.02 ± 0.00</td><td>1226</td><td>1227</td><td>OM</td></tr><tr><td>27</td><td>36.7</td><td>Geraniol</td><td>5.53 ± 0.05</td><td>1234</td><td>1235</td><td>OM</td></tr><tr><td>28</td><td>37</td><td>Linalyl Acetate</td><td>33.01 ± 0.07</td><td>1239</td><td>1239</td><td>OM</td></tr><tr><td>29</td><td>37.6</td><td>Linalyl propionate</td><td>0.08 ± 0.00</td><td>1318</td><td>1319</td><td>OM</td></tr><tr><td>30</td><td>38.5</td><td>Terpenyl acetate</td><td>0.10 ± 0.00</td><td>1334</td><td>1338</td><td>OM</td></tr><tr><td>31</td><td>38.6</td><td>Citronellyl acetate</td><td>0.02 ± 0.00</td><td>1335</td><td>1336</td><td>OM</td></tr><tr><td>32</td><td>40.2</td><td>Neryl acetate</td><td>2.43 ± 0.02</td><td>1342</td><td>1345</td><td>OM</td></tr><tr><td>33</td><td>40.5</td><td>Geranyl acetate</td><td>4.51 ± 0.03</td><td>1359</td><td>1361</td><td>OM</td></tr><tr><td>34</td><td>40.8</td><td>β-Caryophyllene</td><td>0.40 ± 0.01</td><td>1417</td><td>1425</td><td>S</td></tr><tr><td>35</td><td>42</td><td>α-Humulene</td><td>0.05 ± 0.00</td><td>1437</td><td>1440</td><td>S</td></tr><tr><td>36</td><td>42.1</td><td>E-β-Farnesene</td><td>0.02 ± 0.00</td><td>1443</td><td>1444</td><td>S</td></tr><tr><td>37</td><td>44.3</td><td>B-Germacrene</td><td>0.10 ± 0.00</td><td>1475</td><td>1477</td><td>S</td></tr><tr><td>38</td><td>46.9</td><td>β-Bisabolene</td><td>0.02 ± 0.00</td><td>1496</td><td>1499</td><td>S</td></tr><tr><td>39</td><td>57.2</td><td>Nerolidol</td><td>0.15 ± 0.01</td><td>1547</td><td>1550</td><td>OS</td></tr><tr><td>40</td><td>57.8</td><td>Germacra-1,5-dien-4-ol</td><td>0.02 ± 0.00</td><td>1568</td><td>1569</td><td>S</td></tr><tr><td>41</td><td>61.5</td><td>Spathulenol</td><td>0.01 ± 0.00</td><td>1576</td><td>1573</td><td>OS</td></tr><tr><td>42</td><td>63.8</td><td>T-Cadinol</td><td>0.02 ± 0.00</td><td>1626</td><td>1624</td><td>S</td></tr><tr><td>43</td><td>66.8</td><td>α-Cadinol</td><td>0.05 ± 0.00</td><td>1652</td><td>1653</td><td>S</td></tr><tr><td colspan="7">Compound group (%)</td></tr><tr><td colspan="7">Oxygenated Monoterpenes 86.02</td></tr><tr><td colspan="7">Monoterpenes 7.6</td></tr><tr><td colspan="7">Sesquiterpenes 0.47</td></tr><tr><td colspan="7">Oxygenated Sesquiterpenes 0.25</td></tr><tr><td colspan="7">Other 5.29</td></tr><tr><td colspan="7">Total 99.63</td></tr></tbody></table>

A: aldehyde; Alc: aliphatic alcohol; F: Furan; M: Monoterpene; O M: Oxygenated Monoterpene; S: sesquiterpene; O S: Oxygenated sesquiterpene.

The hydrodistillation of the ground leaves of *C. aurantium* resulted in a pale yellowish oil where the EO yield was 0.57%, which was quite similar to the results already reported for this species in Algeria [23]. This yield value was higher than those observed in Tunisian sour orange leaves (0.31–0.56%) collected from different regions and during different seasons [21,22]. This result corresponds to higher values reported in the study of Almeida et al. [38], who examined seven accessions of sour orange from Brazil. This disparity could be explained by environmental and/or genetic factors, particularly for this species, which presents glandular trichomes on the surface of the leaves [25,38–41].

Forty-three constituents make up the sour orange EO. The major components were linalyl acetate, linalool, α-Terpineol, geraniol and geranyl acetate (Table 1). β-Pinene, Neryl acetate, trans-β-Ocimene, β-Myrcene and Nerol were also detected, at least at 2% of total EO (Table 1). These results confirm that the EO of Algerian *C. aurantium* leaves belongs to the linalool/linalyl acetate chemotype, reported to be the most widespread one [21,38].

## 3.2. Phenolic and Antioxidant Activity

Total phenol content (TPC) from sour leaves of Algeria was 3.48 ± 0.10 mg/g (GAE/EO). This value is low compared to results reported in other studies where other organs of *C. aurantium* were analyzed [16,42,43].

It is well known that free radicals cause cell death and tissue damage leading to chronic diseases. Many studies have reported the potential value of using essential oils to eliminate these free radicals [14,15,24,44]. *Citrus* essential oils have been reported to present activities that allow them to fight against cellular damage caused by physiological oxidants. These activities limit the impact of physiological oxidants and free radicals on in vitro assays [19,24,42].

Few studies have evaluated the antioxidant activities of essential oils from *C. aurantium* leaves. We measured the antioxidant activity of essential oils with 2,2-diphenyl-1-
picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) tests, which are simple and widely used for antioxidant studies. These tests were chosen in order to compare our results with results reported in other *Citrus* species [12,16].

Sour orange EO presented a low antioxidant activity, determined by DPPH assays, with IC₅₀ values > 10,000 mg·L⁻¹; meanwhile, ascorbic acid showed a IC₅₀ value of 3.9 mg·L⁻¹. These values were typically low and concur with those reported in Tunisian and Serbian EO of *C. aurantium* [12,16]. In contrast, hydrosol and ethanol extracts from *C. aurantium* from Turkey have shown significant antioxidant activities [18]. Indeed, in this report [18], ethanol and hydrosol were used as solvents, while our study used EO as extracted by hydrodistillation. Moreover, many of the antioxidant activities observed in *C. aurantium* have been assessed in EO extracted from different organs or byproducts of *Citrus* transformation industries. There are no published reports on antioxidant activity of EO from leaves of sour orange. In our study, ABTS radical scavenging assay exhibited low antioxidant activity (38.6 mg Trolox equivalent·g⁻¹). Similar small antioxidant activities of essential oils of numerous plant species against ABTS radicals were detected [44-47]. These discrepancies are mainly due to the small content of phenols in the EO of sour orange [16]. Indeed, the portion of the fruit used for extraction notwithstanding, the major component of sour orange EO displayed no antioxidant effect [12,13,48,49]. This fact is also supported by the low TPC observed in our study. Nevertheless, Hsouna et al. [16] reported an interesting antioxidant activity of EO of sour orange peels. This difference could be due to the EO composition [6]. Phenolic compounds have been shown to serve as electron donors in free radical reactions and seem to be frequently associated with the EO antioxidant effects [18]. The results obtained for sour orange are different from those shown in four *Citrus* species essential oils of peels. Indeed, Lin et al. [24] have reported strong antioxidant activities measured both by DPPH and ABTS tests. Obviously, the chemical composition, environmental conditions as well as genetic background are among the factors which explain these differences.

### 3.3. Collagenase and Elastase Inhibition

Figure 1 shows the results of Collagenase and Elastase inhibition which were represented by anti-collagenase and anti-elastase activities of sour orange EO.

The EO of sour orange leaves is mainly composed of linalool, linalyl acetate and α-terpineol, which constitute more than 73% of the total components (Table 1). These components were reported to exhibit anti-aging properties [50]. Moreover, limonene, which was not present at elevated levels in the study's EO (Table 1), was reported to display higher anti-inflammatory properties [16,51]. In addition to their individual activities, these compounds could display a synergistic action [16,44]. Several Lamiaceae and Apiaceae EOs have been shown to exert anti-aging properties [48-50]. Zemour et al. [38] highlighted the same effect in safflower vegetable oil. All these reports emphasized that the inhibition of collagenase and elastase may spring from the inhibition of the production of pro-inflammatory mediators. Their results mirrored strong anti-aging activities. Even when evaluated using different methods, the unique available work [16] agrees with the findings of the current study. The exploration of skin protective formulations is linked to the use of plant constituents which have an antioxidant activity. This activity is correlated with the ability to protect the different layers of the skin (dermal and epidermal), mainly composed of elastin and collagen. Skin exposure to external aggressions (for example, UV, temperature, etc.) leads to the increase of the enzymes involved in the aging process, including collagenase and elastase. Their activity triggers the degradation of major components such as collagen and elastin. This in its turn accelerates the visible aging of the skin evidenced by age-related skin changes such as wrinkles and sagging skin depending on the exposure to primary risk factors [51,52].
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Collagenase and Elastase inhibition represented by anti-collagenase activity and anti-elastase activity of the EO of *Citrus aurantium* leaves. Means and standard deviation values are displayed. Grey columns represent control and green ones essential oil. Similar letters for the same treatment indicate non-significant differences as indicated by Duncan's multiple range test, at *p* < 0.01.</figcaption></figure>

These results highlight the interest of using the EO of *C. aurantium* leaves as a potential source of valuable components in the pharmaceutical, food and cosmetic industries [6,43]. This is also the case of the EO extracted as a by-product from peels and flowers. Indeed, the EO of sour orange leaves exhibited interesting insecticidal effects against saw-toothed grain beetle and rice weevil [47].

Fungicidal, bactericidal and insecticidal effects were also reported for the EO from leaves, flowers and peels of *C. aurantium* from North Africa and India [11,21,42,53]. In contrast, peel EO demonstrated a larvicidal activity against the Malaria Vector *Anopheles stephensi* [15,47,54]. Moreover, Costa et al. [55] reported an anxiolytic-like activity and LDL-cholesterol lowering effects of mature fruit EO. This effect was reported to be highly effective on patients diagnosed with chronic lymphocytic leukemia who have received EO of *C. aurantium* by inhalation [56]. It is well known that the EO of this species is used to treat gastric disorders [6,57]. Moraes et al. [58] emphasized that, after intraduodenal administration of a single dose of EO of bitter orange to rats, EO may contribute significantly to the development of a remedy for gastric damage prevention.
**4. Conclusions**

Natural compounds, their properties and their activities play a crucial role in maintain-
ing human health and preserving the environment. Given their importance for perfumery,
cosmetic and pharmaceutical uses, EO have attracted interest from researchers. EO from
leaves of C. aurantium grown in Algeria was mostly constituted of linalool, linalyl acetate
and α-Terpineol. EO presented a low phenol content and low antioxidant activity. In
contrast, as a result of this composition, the inhibition of elastase and collagenase activity
was very high. This finding constitutes a first report of anti-elastase and anti-collagenase
activities reported in the EO of leaves from this species, which must be established by con-
ducting more experiments using different methods. If confirmed, the anti-collagenase and
anti-elastase activities of *Citrus aurantium* EO could increase its use in drug formulations
and in pharmaceutical and cosmetic applications.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/agronomy12061466/s1, Figure S1: Chromatogram of C. aurantium essential oil.

**Author Contributions:** Conceptualization, Z.-E.D., H.M.-K. and O.M.; methodology, C.O., B.Z. and O.M.; software, C.O. and Z.-E.D.; validation, Z.-E.D., H.M.-K. and B.Z.; formal analysis, C.O., B.Z. and O.M.; investigation, C.O. and B.Z.; writing—original draft preparation, C.O., B.Z. and Z.-E.D.; writing—review and editing, H.M.-K. and O.M.; supervision, H.M.-K. and Z.-E.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors are grateful to Muriel Cerny from Laboratoire de Chimie Agroin-
dustrielle, Université de Toulouse, France, for her help in samples analyses.

**Conflicts of Interest:** The authors declare no conflict of interest.

**References**

1. Thiviya, P.; Gamage, A.; Piumali, D.; Merah, O.; Madhujith, T. Apiaceae as an Important Source of Antioxidants and Their Applications. *Cosmetics* **2021**, *8*, 111. [CrossRef]

2. Sayed Ahmad, B.; Talou, T.; Saad, Z.; Hijazi, H.; Merah, O. The Apiaceae: Ethnomedicinal family as source for industrial uses. *Ind. Crops Prod.* **2017**, *109*, 661–671. [CrossRef]

3. FAO. *Citrus Fruits: Fresh and Processed*. Statistical Bulletin; FAO: Rome, Italy, 2017; 77p.

4. Hwang, S.-L.; Shih, P.-H.; Yen, G.-C. Neuroprotective effects of Citrus flavonoids. *J. Agric. Food Chem.* **2012**, *60*, 877–885. [CrossRef] [PubMed]

5. Othman, M.; Atiqah, S.N.; Hassan, M.A.; Nahar, L.; Basar, N.; Jamil, S.; Sarker, S.D. Essential oils from the Malaysian Citrus (Rutaceae) medicinal plants. *Medicines* **2016**, *3*, 13. [CrossRef] [PubMed]

6. Bora, H.; Kamle, M.; Mahato, D.K.; Tiwari, P.; Kumar, P. Citrus essential oils (CEOs) and their applications in food: An overview. *Plants* **2020**, *9*, 357. [CrossRef] [PubMed]

7. Tranchida, P.Q.; Bonaccorsi, I.; Dugo, P.; Mondello, L.; Dugo, G. Analysis of Citrus essential oils: State of the art and future perspectives. A review. *Flavour Fragr. J.* **2012**, *27*, 98–123. [CrossRef]

8. De Pasquale, F.; Siragusa, M.; Abbate, L.; Tusa, N.; De Pasquale, C.; Alonzo, G. Characterization of five sour orange clones through molecular markers and leaf essential oils analysis. *Sci. Hortic.* **2006**, *109*, 54–59. [CrossRef]

9. Elshafie, H.S. Plant Essential Oil with Biological Activity. *Plants* **2022**, *11*, 980. [CrossRef]

10. Hosni, K.; Zahed, N.; Chrif, R.; Abid, I.; Medfei, W.; Kallel, K.; Ben Brahim, N.; Sebei, H. Composition of peel essential oils from four selected Tunisian Citrus species: Evidence for the genotypic influence. *Food Chem.* **2010**, *123*, 1098–1104. [CrossRef]

11. Djenane, D. Chemical profile, antibacterial and antioxidant activity of Algerian citrus essential oils and their application in *Sardina pilchardus*. *Foods* **2015**, *4*, 208–228. [CrossRef]

12. Radan, M.; Parcina, A.; Burcul, F. Chemical composition and antioxidant activity of essential oil obtained from bitter orange peel (*Citrus aurantium* L.) using two methods. *Croatica Chem. Acta* **2018**, *91*, 125–128. [CrossRef]

13. Sarrou, E.; Chatzopoulou, P.; Dimassi-Theriou, K.; Therios, I. Volatile constituents and antioxidant activity of peel, flowers and leaf oils of *Citrus aurantium* L. growing in Greece. *Molecules* **2013**, *18*, 10639–10647. [CrossRef] [PubMed]

14. Mohagheghniapoura, A.; Saharkhiza, M.J.; Golmakanic, M.T.; Niakousari, M. Variations in chemical compositions of essential oil from sour orange (*Citrus aurantium* L.) blossoms by different isolation methods. *Sustain. Chem. Pharm.* **2018**, *10*, 118–124. [CrossRef]
15. Sanei-Dehkordi, A.; Sedaghat, M.M.; Vatandoost, H.; Abai, M.R. Chemical compositions of the peel essential oil of *Citrus aurantium* and its natural larvicidal activity against the malaria vector *Anopheles stephensi* (Diptera: Culicidae) in comparison with *Citrus paradisi*. *J. Arthropod-Borne Dis.* **2016**, *10*, 577.

16. Azanchi, T.; Shafaroodi, H.; Asgarpanah, J. Anticonvulsant activity of *Citrus aurantium* blossom essential oil (neroli): Involvement of the GABAergic system. *Nat. Prod. Commun.* **2014**, *9*, 1615–1618.

17. Khodabakhsh, P.; Shafaroodi, H.; Asgarpanah, J. Analgesic and anti-inflammatory activities of *Citrus aurantium* L. blossoms essential oil (neroli): Involvement of the nitric oxide/cyclic-guanosine monophosphate pathway. *J. Nat. Med.* **2015**, *69*, 324–331. [CrossRef]

18. Hsouna, A.B.; Hamdi, N.; Halima, N.B.; Abdelkafi, S. Characterization of essential oil from *Citrus aurantium* L. flowers: Antimicrobial and antioxidant activities. *J. Oleo Sci.* **2013**, *62*, 763–772. [CrossRef]

19. Bnina, E.B.; Hajlaoui, H.; Chaieb, I.; Said, M.B.; Jannet, H.B. Chemical composition, antimicrobial and insecticidal activities of the tunisian *Citrus aurantium* essential oils. *Czech J. Food Sci.* **2019**, *37*, 81–92. [CrossRef]

20. Hosni, K.; Hassen, I.; M'rabet, Y.; Sebei, H.; Casabianca, H. Genetic relationships between some Tunisian *Citrus* species based on their leaf volatile oil constituents. *Biochem. Syst. Ecol.* **2013**, *50*, 65–71. [CrossRef]

21. Ben Hsouna, A.; Gargouri, M.; Dhifi, W.; Ben Saad, R.; Sayahi, N.; Mnif, W.; Saibi, W. Potential anti-inflammatory and antioxidant effects of *Citrus aurantium* essential oil against carbon tetrachloride-mediated hepatotoxicity: A biochemical, molecular and histopathological changes in adult rats. *Environ. Toxicol.* **2019**, *34*, 388–400. [CrossRef]

22. Zarrad, K.; Ben Hamouda, A.; Chaiebb, I.; Laarif, A.; Mediouni-Ben Jemâa, J. Chemical composition, fumigant and anti-acetylcholinesterase activity of the Tunisian *Citrus aurantium* L. essential oils. *Ind. Crop Prod.* **2015**, *76*, 121–127. [CrossRef]

23. Degirmenci, H.; Erkurt, H. Chemical profile and antioxidant potency of *Citrus aurantium* L. flower extracts with antibacterial effect against foodborne pathogens in rice pudding. *LWT Food Sci. Technol.* **2020**, *126*, 109273. [CrossRef]

24. Hamdani, F.Z.; Allem, R. Propriétés antifongiques des huiles essentielles des feuilles de *Citrus* vis-à-vis *d'Alternaria alternata* et *Penicillium* sp in vitro. *Phytothérapie* **2017**, *15*, 263–266. [CrossRef]

25. Lin, X.; Cao, S.; Sun, J.; Lu, D.; Zhong, B.; Chun, J. The chemical compositions, and antibacterial and antioxidant activities of four types of citrus essential oils. *Molecules* **2021**, *26*, 3412. [CrossRef]

26. Proestos, C.; Sereli, D.; Komaitis, M. Determination of phenolic compounds in aromatic plants by RP-HPLC and GC-MS. *Food Chem.* **2006**, *95*, 44–52. [CrossRef]

27. NIST/EPA/NIH. *Mass Spectral Library Gaithersburg*; National Institute of Standard and Technology: Palmer, MA, USA, 2022.

28. Adams, R.P. *Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry*, 4th ed.; Allured Publishing Corp.: Carol Stream, IL, USA, 2007; pp. 102–133.

29. Babushok, V.I.; Linstrom, P.J.; Zenkevich, I.G. Retentions indices for frequently reported compound of plant essential oils. *J. Phys. Chem. Ref. Data* **2011**, *40*, 043101. [CrossRef]

30. Davies, N.W. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and Carbowax 20M phases. *J. Chromatogr.* **A** **1990**, *503*, 1–24. [CrossRef]

31. Dwivedy, A.K.; Prakash, B.; Chanotiya, C.S.; Bisht, D.; Dubey, N.K. Chemically characterized Mentha cardiaca L. essential oil as plant based preservative in view of e_cacy against biodeteriorating fungi of dry fruits, aflatoxin secretion, lipid peroxidation and safety profile assessment. *Food Chem. Toxicol.* **2017**, *106*, 175–184. [CrossRef]

32. Morshedloo, M.R.; Maggi, F.; Neko, H.T.; Aghdam, M.S. Sumac (Rhus coriaria L.) fruit: Essential oil variability in Iranian populations. *Ind. Crops Prod.* **2018**, *111*, 1–7. [CrossRef]

33. Araújo, F.M.; Dantas, M.C.S.M.; Silva, L.S.; Aona, L.Y.S.; de Souza-Neta, L.C. Antibacterial activity and chemical composition of the essential oil of Croton heliotropiifolius Kunth from Amargosa, Bahia, Brazil. *Ind. Crops Prod.* **2017**, *105*, 203–206. [CrossRef]

34. Salachna, P.; Łopusiewicz, Ł.; Wesołowska, A.; Meller, E.; Piechocki, R. Mushroom waste biomass alters the yield, total phenolic content, antioxidant activity and essential oil composition of *Tagetes patula* L. *Ind. Crop Prod.* **2021**, *171*, 113961. [CrossRef]

35. Popovici, C.; Saykova, I.; Tylkowski, B. Evaluation de l'activité antioxydant des composés phénoliques par la réactivité avec le radical libre DPPH. *Rev. Génie Ind.* **2009**, *4*, 25–39.

36. Barragan Ferrer, D.; Venskutonis, P.R.; Talou, T.; Zebib, B.; Barragan Ferrer, M.J.; Merah, O. Bioactive compounds and antioxidant properties of *Myrrhis odorata* deodorized residue leaves extracts from Lithuania and France origins. *Pharm. Chem. J.* **2016**, *3*, 43–48.

37. Wittenauer, J.; Mäckle, S.; Sußmann, D.; Schweiggert-Weisz, U.; Carle, R. Inhibitory effects of polyphenols from grape pomace extract on collagenase and elastase activity. *Fitoterapia* **2015**, *101*, 179–187. [CrossRef]

38. Zemour, K.; Labdelli, A.; Adda, A.; Dellal, A.; Talou, T.; Merah, O. Phenol content, antioxidant and antiaging activities of safflower seed oil (Carthamus tinctorius L.). *Cosmetics* **2019**, *6*, 55. [CrossRef]

39. Almeida, L.A.H.; Santos, J.Z.; Soares Filho, W.S.; Bizzo, H.R.; Silva, J.P.; Vieira, R.F. Chemical Characterization of Leaf Essential Oil from Seven Accessions of Sour Orange (*Citrus aurantium* L.). *J. Essent. Oil Bearing Plant* **2015**, *18*, 426–435. [CrossRef]

40. Ferrer, V.; Costantino, G.; Paoli, M.; Paymal, N.; Quinton, C.; Ollitrault, P.; Tomi, F.; Luro, F. Intercultivar Diversity of Sour Orange (*Citrus aurantium* L.) Based on Genetic Markers, Phenotypic Characteristics, Aromatic Compounds and Sensorial Analysis. *Agronomy* **2021**, *11*, 1084. [CrossRef]

41. Guo, L.; Liu, Y.; Luo, L.; Hussain, S.B.; Bai, Y.; Alam, S.M. Comparative Metabolites and Citrate-Degrading Enzymes Activities in Citrus Fruits Reveal the Role of Balance between ACL and Cyt-ACO in Metabolite Conversions. *Plants* **2020**, *9*, 350. [CrossRef]
42. Dugo, G.; Bonaccorsi, I.; Sciarrone, D.; Costa, R.; Dugo, P.; Mondello, L.; Santi, L.; Fakhry, H.A. Characterization of oils from the fruits leaves and flowers of the bitter orange tree. *J. Essent. Oil Res.* **2011**, *23*, 45–59. [CrossRef]

43. Aazza, S.; Lyoussi, B.; Miguel, M.G. Antioxidant and antiacetylcholinesterase activities of some commercial essential oils and their major compounds. *Molecules* **2011**, *16*, 7672–7690. [CrossRef]

44. Boussaada, O.; Skoula, M.; Kokkalou, E.; Chemli, R. Chemical Variability of Flowers, Leaves, and Peels Oils of Four Sour Orange Provenances. *Essent. Oil Bearing Plant* **2013**, *10*, 453–464. [CrossRef]

45. Mahdi, A.A.; Al-Maqtari, Q.A.; Mohammed, J.K.; Al-Ansi, W.; Cui, H.; Lin, L. Enhancement of antioxidant activity, antifungal activity, and oxidation stability of *Citrus reticulata* essential oil nanocapsules by clove and cinnamon essential oils. *Food Biosci.* **2021**, *43*, 101226. [CrossRef]

46. Rădulescu, M.; Jianu, C.; Lukinich-Gruia, A.T.; Mioc, M.; Mioc, A.; Șoica, C.; Stana, L.G. Chemical composition, in vitro and in silico antioxidant potential of *Melissa officinalis* subsp. officinalis essential oil. *Antioxidants* **2021**, *10*, 1081. [CrossRef] [PubMed]

47. Barragan Ferrer, D.; Venskutonis, P.R.; Talou, T.; Barragan Ferrer, J.M.; Zebib, B.; Merah, O. Identification and in vitro activity of bioactive compounds extracted from *Tussilago farfara* (L.) plant grown in Lithuania and France. *Free. Radic. Antioxid.* **2018**, *8*, 40–47. [CrossRef]

48. Raeis Abad, M.K.; Besheli, B.A. Insecticidal potential of essential oil from the leaves of *Citrus aurantium* L. against *Oryzaephilus surinamensis* (F.), *Lasioderma serricorne* (L.) and *Sitophilus oryzae* (L.). *J. Entomol. Zool. Stud.* **2016**, *4*, 865–869.

49. Perera, S.; Silva, A.B.G.; Amarathunga, Y.; De Silva, S.; Jayatissa, R.; Gamage, A.; Merah, O.; Madhujith, T. Nutritional Composition and Antioxidant Activity of Selected Underutilized Fruits Grown in Sri Lanka. *Agronomy* **2022**, *12*, 1073. [CrossRef]

50. Merah, O.; Sayed-Ahmad, B.; Talou, T.; Saad, Z.; Cerny, M.; Grivot, S.; Evon, P.; Hijazi, A. Biochemical Composition of Cumin Seeds, and Biorefining Study. *Biomolecules* **2020**, *10*, 1054. [CrossRef]

51. Garg, C.; Khurana, P.; Garg, M. Molecular mechanisms of skin photoaging and plant inhibitors. *Inter. J. Green Pharm.* **2017**, *11*, 217–232. [CrossRef]

52. Zhang, S.; Duan, E. Fighting against skin aging: The way from bench to bedside. *Cell Transplan.* **2018**, *27*, 729–738. [CrossRef]

53. El Khetabi, A.; Ezrari, S.; El Ghadraoui, L.; Tahiri, A.; Ait Haddou, L.; Belabess, Z.; Merah, O.; Lahlali, R. In Vitro and In Vivo Antifungal Activities of Nine Commercial Essential Oils against Brown Rot in Apples. *Horticulturae* **2021**, *7*, 545. [CrossRef]

54. El-Akhal, F.; El Ouali Lalami, A.; Guemmouh, R. Larvicidal activity of essential oils of *Citrus sinensis* and *Citrus aurantium* (Rutaceae) cultivated in Morocco against the malaria vector *Anopheles labranchiae* (*Diptera: Culicidae*). *Asian Pac. J. Trop. Dis.* **2015**, *5*, 458–462. [CrossRef]

55. Costa, C.A.R.A.; Cury, T.C.; Cassettari, B.O.; Takahira, R.K.; Flório, J.C.; Costa, M. *Citrus aurantium* L. essential oil exhibits anxiolyticlike activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment. *BMC Complementary Altern. Med.* **2013**, *13*, 42. [CrossRef] [PubMed]

56. Fernandes Pimenta, F.C.; Alves, M.F.; Fernandes Pimenta, M.B.; Melo, S.A.L.; Figueirêdo de Almeida, A.A.; Leite, J.R.; de Morais Pordeus, L.C.; Melo Diniz, M.F.F.; de Almeida, R.N. Anxiolytic Effect of *Citrus aurantium* L. on Patients with Chronic Myeloid Leukemia. *Phytother. Res.* **2016**, *30*, 613–617. [CrossRef] [PubMed]

57. Rozza, A.L.; Pellizzon, C.H. Essential oils from medicinal and aromatic plants: A review of the gastroprotective and ulcer-healing activities. *Fundam. Clin. Pharm.* **2013**, *27*, 51–63. [CrossRef]

58. Moraes, T.M.; Kushima, H.; Moleiro, F.C.; Santos, R.C.; Rocha, L.R.; Marques, M.O.; Vilegas, W.; Hiruma-Lima, C.A. Effects of limonene and essential oil from *Citrus aurantium* on gastric mucosa: Role of prostaglandins and gastric mucus secretion. *Chem.-Biol. Inter.* **2009**, *180*, 499–505. [CrossRef]