EJBR2021v11i4art501-508 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2021; 11(4): 501-508 DOI: http://dx.doi.org/10.5281/zenodo.5552960 Organ dependency variation of the chemical composition of Ziziphus lotus volatile fractions Touka Letaief 1,2,3,*, Stefania Garzoli 4, Elisa Ovidi 3, Antonio Tiezzi 3, Chokri Jeribi 1, Manef Abderrabba 1, Jamel Mejri 1 1 Laboratory of Materials Molecules and Applications (LMMA), IPEST, BP 51, 2070 La Marsa, Tunis, Tunisia 2 National Agronomic Institute of Tunisia (INAT), University of Carthage, Tunis, Tunisia 3 Department for the Innovation in Biological, Agrofood and Forestal Systems, Tuscia University Viterbo, Italy 4 Department of Drug Chemistry and Technology, Sapienza University, Rome, Italy * Corresponding author e-mail: touka.letaief@gmail.com Received: 30 July 2021; Revised submission: 21 August 2021; Accepted: 06 September 2021 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2021. Licensee Joanna Bródka, Poland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: The extended application fields of the essential oils keep them a subject of interest. In this study, we investigated the aerial part essential oil and the fruit essential oil of the wild plant Ziziphus lotus, collected from the southern region of Tunisia. These essential oils obtained by hydrodistillation using a Clevenger-type apparatus showed an extraction yield of 0.013% and 0.0046% respectively. The qualitative and quantitative analysis of the samples using GC-MS/GC-FID revealed two distinct compositions. Apocarotenoid derivatives characterized the essential oil of the aerial part; the major compound was hexahydrofarnesyl acetone (23.2%) followed by geranylacetone (12.5%) and cis-hexenyl-3-benzoate (11.1%). While the abundance of fatty acid marked the fruit essential oil. The noticed major compounds were 2-pentadecanone (16.9%), dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%). Such chemical composition may explain the traditional use of Ziziphus lotus as a drug to treat various pathologies. Keywords: Ziziphus lotus; Essential oil; Apocarotenoid; Hexahydrofarnesyl acetone; Fatty acids; GC- MS/GC-FID. 1. INTRODUCTION Due to their safety, plant extracts are gaining an increasing interest in several fields. They are, in fact, used as a flavoring and functional agent in food industries, as a substitute for many chemical compounds in drugs formulation as well as preservative factors in cosmetics products [1-4]. Thus, the discovery of new plant molecules may bring an unexpected endowment. The genus Ziziphus, which belongs to the Rhamnaceae family, is widely distributed in many regions all over the world. For instance, it is common in Asia, Africa, North America, South America, Oceania and Europe [5]. This group is represented by more than 200 species [6], from which only three species can be encountered in Tunisia: Z. spina-christi (L.) Willd, Z. vulgaris Lam. and Z. lotus (L.) Lam. [7]. In our study, we have interested in the Tunisian indigenous species, Ziziphus lotus which reveals a high capacity for adaptation under different environmental circumstances. Z. lotus is a source of several classes of natural products endowed of numerous biological activities. The leaves of this plant are rich in saponins, linolenic acid (18:3n-3), vitamin C, vitamin E, flavonoids, Letaief et al. Organ dependency and chemical composition of Ziziphus lotus 502 European Journal of Biological Research 2021; 11(4): 501-508 tannins [8-10]. The roots are a source of cyclopeptide alkaloids, saponins, vitamin C, polyphenol and essential fatty acids [9,11-13] and the fruits are characterized by their content in sterols, fatty acid, vitamin A, vitamin C, polyphenols, flavonoids, polysaccharides [9-15]. The large content of natural products confers to Z. lotus many biological activities. For instance, the leaves are known for anti-inflammatory, analgesic, antidiabetic, and antiulcerogenic properties [12,14,16]. The root extracts prove many activities such as antioxidant, anti- inflammatory, analgesic, anti-spasmodic and antidiabetic [14,17,18] and the fruits show antioxidant, gastroprotective, antibacterial and immunomodulatory properties [9,19,20]. According to literature, only one study was found to be focusing on Z lotus fruit essential oil [21]. Hence, since this limited investigations, this work aimed to study the aerial part essential oil and the fruit essential oil obtained from Z. lotus by hydrodistillation and to determine its compositions by GC-MS/GC- FID. 2. MATERIALS AND METHODS 2.1. Plant material From a deserted region in the village of Oudhref-Gabes situated in the south of Tunisia, the samples of Z. lotus were collected. During the flowering stage in May 2017, the aerial part samples were collected and during the summer in August 2017 the fruit was harvested. Samples were shade dried for two weeks at room temperature and then stored at the absence of light and under dry conditions until use. The plant identification was carried out by Professor Mohamed Boussaid (Department of Biology, Laboratory of Plant Biotechnology, National Institute of Applied Science and Technology). 2.2. Essential oils extraction 1 kg of the aerial part of Z. lotus (leaves and flowers) was crushed. The obtained powder was mixed with 4 L of water in a 10 L flask and subjected to the stem distillation in a Clevenger-type apparatus. The dried fruits were pitted, and then 50 g of the edible part was soaked in 500 mL of water in a 1 L flask. After 4 hours of hydrodistillation, the essential oils were collected. The obtained essential oils were dried and kept at 4°C for analysis. 2.3. Chemical composition analysis Chemical analysis of Z. lotus essential oils was performed by a Turbo-mass Clarus 500 GC-MS/GC- FID from Perkin Elmer instruments (Waltham, MA, USA) equipped with a Stabil wax fused-silica capillary column (Restek, Bellefonte, PA, USA) (60 m × 0.25 mm, 0.25 µ m film thickness). As operating conditions used, GC oven temperature was kept at 60°C for 5 min and programmed to 220°C at a rate of 5°C/min, and kept constant at 220°C for 25 min. Helium was used as carrier gas at a flow rate of 1 mL/min. Solvent delay was 0–2 min and scan time was 0.2 s. Mass range was from 30 to 350 m/z using electron-impact at 70 eV mode. 2 μl of Z. lotus essential oil was diluted in 1 ml of methanol and 1 μl of the solution was injected into the GC injector at the temperature of 280°C. The analysis was repeated twice. Relative percentages for quantification of the components were calculated by electronic integration of the GC-FID peak areas. The identification of the constituents was made by comparing the obtained mass spectra for each component with those reported in mass spectra Nist and Willey libraries. Linear retention indices (LRI) of each compound were calculated using a mixture of aliphatic hydrocarbons (C8-C30, Ultrasci) injected directly into GC injector at the same temperature program reported above. Letaief et al. Organ dependency and chemical composition of Ziziphus lotus 503 European Journal of Biological Research 2021; 11(4): 501-508 3. RESULTS AND DISCUSSION The hydrodistillation of Z. lotus aerial part, as well as Z. lotus fruit, allowed collecting two whitish essential oils with an extraction yields of 0.013% (w/w) and 0,0046% respectively. No previous investigations were found about the essential oil composition of Z. lotus aerial part. Yet, concerning the fruit essential oil, the extraction yield is in concordance with the yield reported by Widad et al. (0.005%) [21]. The list of compounds, their percentages as well as their retention indices are reported in Table 1. Table 1. Chemical composition of Ziziphus lotus essential oils using GC-MS/GC-FID. Component LRI1 LRIlit 2 (%) in recovered essential oil AP EO F EO Nonanal 1390 1408 0.2 - Decanal 1497 1511 0.3 - Linalool 1547 1553 0.1 - 2-Undecanone 1610 1606 2.9 - β-Cyclocitral 1619 1622 0.5 - L-α-terpineol 1685 1690 + 0.3 - α-Farnesene 1720 1725 0.4 - Carvone 1732 1740 0.9 - δ-Cadinene 1741 1749+ 0.7 - Tridecanal 1812 1821 1.9 - Damascenone 1820 1827 1.3 - Geranylacetone 1872 1877 12.5 - α-Calacorene 1910 1916 3.0 - Trans-β-ionone 1938 1956 6.0 - D-nerolidol 1984 2011 1.9 - E-nerolidol 1991 2013 5.9 - Hexyl-benzoate 2030 2033 3.1 - Hexahydrofarnesyl acetone 2114 2118 23.2 2.9 Cis-hexenyl -3-benzoate 2120 2123 11.1 - Cadalene 2195 2200 0.1 - α-Cadinol 2211 2218 0.9 - Azulol 2220 * 2.8 - Farnesyl acetone 2360 2363 4.6 - Dodecanoic acid 2474 2479 7.4 5.9 Tetradecanoic acid 2679 2716 6.8 5.1 Decanoic acid, ethyl ester 1612 1614 - 3.6 Undecanoic acid, ethyl ester 1732 1737 - 2.4 2-tridecanone 1800 1803 - 0.4 Dodecanoic acid, ethyl ester 1850 1856 - 14.5 Ethyl tridecanoate 1944 1943 - 0.6 2-pentadecanone 2026 2028 - 16.9 Tetradecanoic acid, ethyl ester 2055 2059 - 5.0 Ledol 2061 2060 - 1.3 Pentadecanoic acid, ethyl ester 2178 2179 - 6.3 Hexadecanoic acid, ethyl ester 2247 2246 - 3.4 Letaief et al. Organ dependency and chemical composition of Ziziphus lotus 504 European Journal of Biological Research 2021; 11(4): 501-508 Component LRI1 LRIlit 2 (%) in recovered essential oil AP EO F EO Ethyl-9-hexadecanoate 2290 2288 - 1.2 n-Decanoic acid 2305 2300 - 8.2 Undecanoic acid 2402 2400 - 0.7 Ethyl oleate 2781 2480 - 1.8 Tridecanoic acid 2600 2603 - 1.0 13-Epimanool 2670 2676* - 5.2 n-Hexadecanoic acid 2943 2946 - 13.0 Total 98.8 99.4 1 Linear Retention indices measured on polar column; 2 Linear Retention indices from literature; *LRIlit not available; +Normal alkane RI; AP-EO: Aerial Part Essential oil; F-EO: Fruit Essential oil. The purpose of this study was to identify the essential oil composition of the different part of the desertic plant Z. lotus. Thus, as summarized in Table 1, twenty-five compounds were identified for AP-EO and twenty compounds for F-EO accounted for 98.8% and 99.4% of the total volatile fractions respectively. Each EO presents a specific composition mostly different to the other EO. Hence, only hexahydrofarnesyl acetone, dodecanoic acid and tetradecanoic acid are showed as the common compounds in both EO. In fact for the AP-EO, twenty-five compounds were identified by GC-MS/GC-FID. The three major compounds were hexahydrofarnesyl acetone (23.2%), geranylacetone (12.5%) and cis-hexenyl -3-benzoate (11.1%). The percentage of each other compound is less than 10%. The components of this EO can be grouped as follow; apocarotenoid derivatives (47.74%), ester (14.18%), saturated fatty acid (14.16%), oxygenated sesquiterpene (8.71%), sesquiterpene hydrocarbons (7.05%), oxygenated monoterpenes (2.96%) and others (4.99%) (Fig. 1). Figure 1. Grouped components of Ziziphus lotus aerial part essential oil (%). The apocarotenoids which characterize AP-EO are mainly generated from carotenoids by a specific cleavage dioxygenase (CCD) along the polyene double bonds [22]. These isoprenoids, known for their volatility in comparison with carotenoids, are necessary for both primary and secondary plant metabolism and have many benefits for human and animal health [22, 23]. Letaief et al. Organ dependency and chemical composition of Ziziphus lotus 505 European Journal of Biological Research 2021; 11(4): 501-508 In our case all the apocarotenoids are ketones and among such apocarotenoids, hexahydrofarnesyl acetone is the main compound with 23.22%. Examining the composition of the essential oil of other species of ziziphus, hexahydrofarnesyl acetone was found as the common compound; its concentration seems to be dependent by both organs and species. In fact, it is about 9.1% in the aerial part essential oil of Zizyphus jujuba and in trace in the fruit essential oil of Ziziphus spina-christi [24]. 6,10,14-Trimethylpentadecan-2-one (also known as Hexahydrofarnesyl acetone; HHA) is a derivative of the diterpene alcohol, phytol, and was found to be a major component in tibial fragrances of male orchid bees, Euglossa spp. [25]. HHA is a chiral molecule with four possible stereoisomers, (6R, 10R)-, (6R, 10S)-, (6S, 10R)-, and (6S,10S)-6,10,14- trimethylpentadecan-2-one. With a molecular weight of 268, it is considered as the largest natural molecule known to attract male orchid bees in pure form. HHA is relatively widespread among the many floral scents and essential oils [26] even if only as a minor component. Notable exceptions are a small number of euglossophilous orchids, in which HHA is the dominant compound found in the floral headspace [27,28]. This compound is known as a potent antimicrobial agent against Gram-positive and Gram-negative bacteria [29]. Also, it is judged as a phytotoxic agent [30]. The two-second major compounds in this apocarotenoid group are a C13 ketone; geranylacetone (12.5%) and trans-beta-ionone (6.0%). These composites are known as flavoring agents. As reported by Ghannadi et al. [17], geranylacetone is found to be the most aboundant compound in the leaf essential oil of Ziziphus spina Christi (14.0%). Geranylacetone is a constituent of many essential oils including peppermint (Mentha piperita) and Carolina vanilla (Carphephorus odoratissimus). It belongs to the class of organic compounds known as acyclic monoterpenoids. Geranylacetone exhibits olfactory, germicidal and antimicrobial properties. Twelve E/Z-mixtures of analogues of geranylacetone was examined for its odor and antimicrobial activity [31]. The ester group is constituted by cis-hexenyl-3-benzoate (11.1%) and a little quantity of hexyl benzoate (3.08%). Cis-hexenyl-3-benzoate is a flavouring agent present in many fruits. In our sample, dodecanoic acid (7.4 %) and tetradecanoic acid (6.8%) are the two saturated fatty acids. A low percentage of sesquiterpene is presented and it is about 8.71% of the oxygenated one, mainly represented by E-nerolidol, and about 7.05% of hydrocarbons one. Monoterpene hydrocarbons were completely absent, while oxygenated monoterpenes reached a low level (2.96%). Concerning Z. lotus fruit essential oil, the major compound was the oxygenated sesquiterpene 2-pentadecanone (16.9%), followed by two saturated fatty acids: dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%). Fatty acid represents 69.1 % of the total essential oil, among them unsaturated fatty acid represents only 3.0%. Oxygenated sesquiterpene represents 19.0% (Fig. 2). Figure 2. Grouped components of Ziziphus lotus fruit essential oil (%). Letaief et al. Organ dependency and chemical composition of Ziziphus lotus 506 European Journal of Biological Research 2021; 11(4): 501-508 With 69.1%, fatty acid remains the major group, extended from C11 to C20. Those results are quantitatively quite similar to those illustrated by Widad et al. [21], where the percentage of fatty acid is about 78.9%. However, in term of quality, the composition is different. Thus, dodecanoic acid ethyl ester (14.5%) and n-hexadecanoic acid (13.0%) represent the major compounds in the fatty acid group of our sample, yet in the named study [21], ethyl hexadecanoate (12.0%) and decanoic acid (11.0%) are the main ones. In this current study, the essential oil was obtained by the hydrodistillation of the edible part of the fruit nevertheless in the study of Widad et al. [21] the essential oil was obtained by the hydrodistillation of the whole fruit, which may explain this difference. The major oxygenated sesquiterpene is 2-pentadecanone (16.9%) known as a flavoring ingredient. Hence, these results proved a noticeable difference, in terms of quality and quantity, of volatile compounds depending on plant organs. It is worth to note that the study of Z. lotus essential oil is limited. In fact, to the best of our knowledge, this is the first study dealing with Z. lotus aerial part essential oil. 4. CONCLUSION The hydrodistillation of Z. lotus organs (the aerial part and the fruit) allowed collecting two whitish essential oils. The qualitative and quantitative analysis carried out by GC-MS/GC-FID showed the richness of AP-EO in apocarotenoid compounds such as hexahydrofarnesyl acetone (23.22%) and geranylacetone (12.55%), while the F-EO was characterized by the abundance of fatty acids with 69.1%. Such compounds are presently tested in our laboratories for evaluation of antibacterial and cytotoxic properties on bacteria and mammal cells. 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