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Introduction

Cyperus rotundus is a member of Cyperaceae family widely distributed in tropical, subtropical, and temperate regions of the world. This plant grows well in almost any soil type, over a wide range of soil humidity, pH, and altitude.It is annual plant that produces prominent swollen underground tuberous bases that remain dormant after the growing season and under adverse conditions [1-3]. Cyperus rotundus is a versatile plant, widely used in traditional medicine around the world and as raw material for perfumes [4-7]. C. rotundus rhizome essential oil possessed antioxidant [6,8,9], antibacterial [6,8-14], insecticide [2,15,16],antimutagenic [6,17], anti-inflammatory [7], antiarthritic [7], analgesic [7] and anticonvulsant activities [7].

C. rotundus tubers show great variation in essential oil composition and several chemotypes have been described.Cyperene (19.2-30.9%) and α-cyperone (4.5-25.2%) were the most abundant constituents in the oils of species from Nigeria and Tunisia [17,18]. Chinese, Iranian and Turkish authors have also reported this high content of cyperene. In China, the essential oil was dominated bycyperene (41.03%), β-caryophyllene (5.32%), α-selinene (4.37%) and α-copaene (4.36%)[19]. In Iran the chemotype was cyperene (16.9%), caryophyllene oxide (8.9%), α-longipinan (8.4%) and β-selinene (6.6%) [20]. In Turkey, cyperene(30.5% and 28.0%), α-copaene (10.6% and 12%) and α-ylangene (7.7% and 10.5%) were identified as the main volatile components of rhizomes [21]. Another study conducted in Iran revealed the presence of elemenone (13.59%), α-cyperone (13.14%), and caryophyllene oxide (13.03%) [15]. The Brazilian species contained α-cyperone (22.8%) and cyperotundone (12.1%) as the main in the essential oil compounds [22]. α-Cyperone (21.1%) has also been reported as the main compound of essential oil from Saudi Arabia along with 4-oxo-α-ylangene (12.8%) [2]. Cyperotundone was also present in the essential oil from Iraq which was mainly composed of two compounds namely cyperene (37.9%) and cyperotundone (11.2%) [23]. The rhizome oils of C. rotundus from India consisted mainly of α-copaene (11.4-12.1%), cyperene (8.04-11.07%), valerenal (8.07-9.08%) and caryophyllene oxide (7.08-9.07%) [24]. Sonwa and Koenig (2001) studied the essential oil of C. rotundus from Germany and found that it was dominated by cyprotene, α-copaene, cyperene,α-selinene and rotundene [25]. More recently, Cisse etal (2021) reported this humulene epoxide 2 (26.1%), caryophyllene oxide (19.2%), longiverbenone (11.3%) chemotype in the essential oil of C. rotundus collected in Senegal [26].

The purpose of this study was to determine the chemical composition of C. rotundus oils collected in different areas in Senegal and to compare this with those of essential oils obtained from other geographical region.

Material and Methods

Plant material

Thirty C. rotundus rhizome samples were collected from five localities in Senegal given in Figure 1: Mont-Rolland (14°55.5988’N, 16°58.59879’W; six samples: M1-M6), Ndiayene Pendao (16°30.24223’N, 15°3.17579’W; seven samples: N1-N7), Guede (16°32.54537’N, 14°45.15816’W; six samples: G1-G6), Ngano (15°16.5628’N,13°2’.52007’W; five samples: NG1-NG5) and Ziguinchor (12°34’336’N, 16°17’35879’W; six samples: Z1-Z6).Each rhizome sample is taken from an area of approximately 200m2. The sampling areas are at least 500 meters apart. The plant material was identified by the technicians from the Botany department of the Fundamental Institute of Black Africa (IFAN) at Cheikh Anta Diop University in Dakar.

Extraction of Essential Oils

Plant samples were air dried for a period of four weeks at room temperature. The plant material was powdered with an average particle size of 0.2 mm using a blade grinder (Polymix PX-MFC 90D, KINEMATICA AG, Luzern,Switzerland). The samples were hydrodistilled (6 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia [27].

Chemical Compositions

The chromatographic analyses were carried out using a Perkin-Elmer Autosystem XL GC apparatus (Walthon, MA, USA) equipped with dual flame ionization detection (FID) system and fused-silica capillary columns, namely, Rtx-1 (polydimethylsiloxane) and Rtx-wax (poly- ethyleneglycol) (60 m × 0.22 mm i.d; film thickness 0.25μm). The oven temperature was programmed from 60 to 230°C at 2°C/min and then held isothermally at 230°C for 35 min: hydrogen was employed as carrier gas (1mL/min).The injector and detector temperatures were maintained at 280°C, and samples were injected (0.2 μL of pure oil) in split mode (1:50). The retention indices (RI) of the compounds were determined relative to the retention times of a series of n-alkanes (C5–C30) by linear interpolation using the equation of Van den Dool and Kratz (1963) with the aid of the Perkin-Elmer software (Total Chrom navigator). The relative percentages of the oil constituents were calculated from the GC peak areas, without applying of correction factors.

The samples were also analysed with a Perkin-Elmer Turbo mass detector (quadrupole) coupled to a Perkin-ElmerAutosystem XL, equipped with fused-silica capillary columns Rtx-1 and Rtx-Wax. The oven temperature was programmed from 60 to 230°C at 2°C/min and then held isothermally at 230°C (35 min): hydrogen was used as carrier gas (1 mL/min). The following chromatographic conditions were employed: injection volume, 0.2 μL of pure oil; injector temperature, 280°C; split, 1:80; ion source temperature, 150°C; ionization energy, 70 eV; MS (EI) acquired over the mass range, 35–350 Da; scan rate, 1 s.Identification of the components was based on: (a) comparison of their GC retention indices (RI) on non-polar and polar columns, determined from the retention times of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data; (b) on computer matching with commercial mass spectral libraries [28-30] and comparison of spectra with those of our personal library; and (c) comparison of RI and MS spectral data of authentic compounds or literature data.

Statistical Analysis

Data analyses were performed using PCA and CA.Both methods aim at reducing the multivariate space in which objects (oil samples) are distributed, but are complementary in their ability to present results. Indeed, PCA provides the data for plots in which both objects (oil samples) and variables (oil components) are represented, while cluster analysis (CA) informs a classification tree in which objects (sample locations) are gathered. The PCA was carried out using the function PCA of the statistical XLSTAT software (Addinsoft, Paris). The discriminate variables (volatile components) have been selected using function of the statistical software. A dendrogram was produced by CA using Ward’s method of hierarchical clustering, based on Euclidean distances between pairs of oil samples.

Results and Discussion

Yields of essential oils given in Table 1 were calculated on the basis of the mass of dry plant matter. They are between 0.6 and 2.6% (Mean ± SD: 1.19 ± 0.53%). These results are consistent with literature reports indicating yields of 0.20–2.60% [2,6,8-11,14,16,18-20,23,31,32].

The analysis of the rhizome essential oils by GC/-FID and GC/MS allowed the identification of 49 compounds accounting for 81.0 to 94.0% of the total compositions (Table 1). These essential oils mainly consisted of high amounts of sesquiterpenes. However, the main compounds varied considerably from one sample to another: humulene epoxide 2 (7.0-38.3%), caryophyllene oxide (5.5-28.1%), longiverbenone (3.0-9.4%), cyperotundone (1.2-15.4%), cyperene (0.7-11.1%) and mustakone (1.0-9.1%) (Figure 2). Thus, statistical analyses were performed on the 30 oil compositions to highlight the chemical variability. Figure 3 was obtained from the correlation matrix calculated with the standardized matrix. As shown in Figure 3, the principal factorial plane (constructed with axes 1 and 2) summarizes 78.57% of the entire variability of essential oils. The distribution of oil samples from C. rotundus oils is reported in Figure3a. The distribution of variables (volatile components) is shown in Figure 3b (11 variables). The graph drawn using PCA suggested the existence of two main groups of essential oils (Figure 3a). Statistical analysis using CA was also used to show the correlation between the chemotypes and the geographical distribution of samples. The dendrogram obtained by cluster analysis (CA) reinforced the clustering observed using PCA by grouping the 30 oil samples from C. articulatus into the same two main clusters (Figure 4).

The first group included the samples collected in Ngano (NG) and Ziguinchor (Z). The dominant compounds in this group were of humulene epoxide 2, caryophyllene oxide, mustakone and cyperotundone. This group was divided into two subgroups. The first subgroup, including Ngano samples, showed a high content of humulene epoxide 2 (21.9-23.5%, mean: 22.6%, SD: 0.6%) and cyperotundone(10.2-14.0%, mean: 11.5%, SD: 1.5%). Instead, the high content of mustakone and cyperotundone was a feature of the second subgroup, which included Ngano samples.To our knowledge, these chemotypes have never been described in the literature. However, the presence of mustakone and cyperotundone at significant levels has been reported in a study conducted in Tunisia [8,17]. Cyperotundone has also been described in essential oils of C. rotundus in Asia [32], Iran [20], Marocco [12] and Brazil [22].

The second group includes specimens collected from Mont-Rollant (M), Ndiayene Pendao (N) and Guede (G), which have a high percentage of caryophyllene oxide (18.0-28.1%, mean: 23.2%, SD: 2.9%), humulene epoxide 2(26.4-38.3, mean: 32.4%, SD: 3.3%). Similarly, high contents of humulene epoxide 2 and caryophyllene oxide have been reported for C. rotundus oil from Louga region in Senegal.However, these compounds were at low levels or absent in essential oils of C. rotundus from Iran [20], India [24] and Brazil [24].

In summary, the results obtained from the analysis of the thirty samples have led to the establishment of three chemotypes to C. rotundus essential oils. These results are based on single samples to each collection site and do not account for within site variation. However, the chemical composition of the analyzed essential oils showed qualitative and quantitative variation by the influence of the localization.

Conclusion

The present investigation demonstrated the important of chemical variability of the essential oils isolated from the rhizomes of C. rotundus populations growing wild in Senegal. Principal component analysis (PCA) and cluster analysis (CA) (dendrogram) applied on the matrix linking essential oil compositions and sample locations allowed the distinction of two groups within the oil samples. The dominant compounds in the first group were humulene epoxide2, caryophyllene oxide, mustakone and cyperotundone and those in the second group were caryophyllene oxide and humulene epoxide 2. To our knowledge, we have reported the first chemotype for the first time. In perspective, we will evaluate their biological activities and also produce cosmetic formulations based on these essential oils.