Chemical composition, antioxidant, anti- inflammatory and antiproliferative activities of the essential oil of Cymbopogon nardus, a plant used in traditional medicine

Objectives: Natural products commonly used in traditional medicine, such as essential oils (EOs), are attractive sources for the development of molecules with anti-proliferative activities for future treatment of human cancers, e.g., prostate and cervical cancer. In this study, the chemical composition of the EO from Cymbopogon nardus was characterized, as well as its antioxidative properties and anti-inflammatory and antiproliferative activities on LNCaP cells derived from prostate cancer. Methods: The chemical composition of the EO was determined by GC/FID and GC/MS analyses. The antioxidative properties were assessed using DPPH radical scavenging assay and ABTS+• radical cation decolorization assay, and the anti-inflammatory capacity was determined by the inhibition of the lipoxygenase activity. Antiproliferative activity was evaluated by MTT assay. Results: Collectively, our data show that the major constituents of C. nardus EO are citronellal (33.06 %), geraniol (28.40 %), nerol (10.94 %), elemol (5.25 %) and delta-elemene (4.09 %). C. nardus EO shows modest antioxidant and anti-inflammatory activity compared to the standard galic acid. C. nardus EO exhibits the best antiproliferative activity on the prostate cancer cell line LNCaP with an IC50 of 58.0 ± 7.9 μg/mL, acting through the induction of the cell cycle arrest. Conclusions: This study has determined that C. nardus EO efficiently triggers cytotoxicity and pens a new field of investigation regarding the putative use of this EO in vivo.


Background
Cancer continues to be a major public health problem worldwide [1]. It is the second leading cause of morbidity, globally, with about 8.8 million deaths in 2015 [2]. Cervical cancer is the fourth most common cancer in women and the fourth leading cause of cancer death in women [3].
Prostate cancer is the second most common malignancy in men worldwide, and its incidence is increasing. The development of better strategies to prevent and to improve access to care and treatment in developing countries is mandatory. Interestingly, plants have been at the origin of important anti-cancer molecules such as paclitaxel, docetaxel, vinblastine, and vincristine [4][5][6][7]. Thus, isolated molecules from plants remain a critical area in the field of drug development.
C. nardus is particularly known for its antioxidant, anti-inflammatory, and antimicrobial properties [21][22][23]. The purpose of this work was to determine the chemical composition, chemotype and to analyse the anti-radical and anti-proliferative activity of the EO of C. nardus, used in Burkina Faso (West Africa), for the potential treatment of inflammatory and oxidative diseases. Fractions of fresh plant material (1 Kg) were submitted to hydrodistillation using an alembic/Clevenger-type apparatus for 3 h, as described previously [14]. EOs were stored in airtight containers in a refrigerator at 4 o C until GC-FID and GC/MS analyses and biological tests. EOs were diluted in hexane (1/30, v/v) for GC/FID analysis.

Chemical composition Gas chromatography-flame ionization detector (GC/FID) analysis
The composition of the EO was determined as previously described [15]. Briefly, gas chromatography of hexane diluted EO was performed on an Agilent gas chromatograph model 6890 (Agilent, Palo Alto, Ca), equipped with a 30m x 0.25 mm, 0.25 µm film thickness column under a hydrogen flow, from 50 o C (5 min) to 300 o C with an increasing temperature of 5 o C/min. Samples were injected in split mode, with injector and detector temperatures at 280 and 300 o C, respectively [14].

Gas chromatography-mass spectrometry (GC/MS) analysis
Mass spectrometry analyses have been reported previously [15]. Briefly, an Agilent gas chromatograph model 7890 coupled to an Agilent MS model 5975 was used. Helium was used with an average flow of 1.0 mL/min. The oven temperature program was from 50 o C (3.2 min) to 300 o C at 8 o C/min, 5 min post-run at 300 o C. The sample was injected in split mode, injector and detector temperatures at 250 o C and 280 o C, respectively [14]. The MS working in electron impact mode at 70eV; electron multiplier, 1500 V; ion source temperature, 230 o C; mass spectra data were acquired in the scan mode in m/z range 33-450 [14].

Identification of components
The main compounds present in the EO of C. nardus have been identified as previously described [15]. Using compound standards to identify EO components would have been the preferred state-of-the-art methodology, however, due to technical and resource constraints [14], we performed retention indices and comparisons using the NIST library [24] or literature [25]. Component relative percentages were calculated based on GC peak areas without using correction factors [14].

Antioxidant activity DPPH radical scavenging assay
DPPH (Sigma-Aldrich, L'Ile d'Abeau, France) radical scavenging activity was measured as described by Velasquez [30]. Briefly, the EO of C. nardus at 8.8 mg/ mL was first serially diluted in a 96-well plate. Then, 100 µL of each EO concentration was mixed with 100 µL of DPPH (30 mg/L in methanol). After 30 min of incubation in the dark, the absorbance was read at 517 nm using a UV/Visible spectrophotometer. Gallic acid was used as a control. The radical scavenging activity was expressed as a percentage inhibition according to the formula: according to the formula: Radical scavenging capacity (%) = bsorbance Blank − Absorbance Sample Absorbance Blank 100 The concentrations were expressed in μg of extracts/μg of DPPH by the formula:

Concentration = Mass of DPPH
The concentrations were expressed in µg of extracts/µg of DPPH by the formula: The concentration of extract capable of scavenging 50% of the DPPH radicals was then determined graphically.

ABTS+• radical cation decolorization assay
The spectrophotometric analysis of ABTS+• scavenging activity was determined according to the method of Re et al. [31]. Briefly, the ABTS+• solution was prepared of by dissolving 10 mg of ABTS+• in 2.6 ml of distilled water. Then, 1.7212 mg of potassium persulfate was added, and the mixture was kept in the dark at room temperature for 12 h. The mixture was then diluted with ethanol in order to obtain an absorbance of 0.70 ± 0.02 to 734 nm.
In 96-well plates, 50 µl of ethanolic extract solution at an initial concentration of 4.4 mg/mL was added to 200 µl of freshly prepared ABTS+• solution. The same process was carried out for gallic acid at an initial concentration of 1.25 mg/mL and used as a standard. The mixture made in the 96-well plates was then incubated in the dark at room temperature for 15 min, and the absorbance was read at 734 nm against a standard curve of 5,7,8-tetramethyl-2-carboxylic acid 6-hydroxy-2 (Trolox, Sigma-Aldrich) using a spectrophotometer. The activity of the EO from C. nardus on the radical cation ABTS+• was expressed in micromolar Trolox equivalent per gram of EO (µM TE/g) using the following formula: C = (cx D) / Ci. C, the concentration of the EO from C. nardus in µM TE/g; c, the concentration of the sample read; D, the dilution factor and Ci, the concentration of the stock solution.

Anti-inflammatory capacity
Lipoxygenase (EC 1.13.11.12) type I-B inhibiting activity was assayed spectrophotometrically as described by Lyckander and Malterud [32] with minor modifications. Briefly, the EO from C. nardus was initially diluted in the 96-well plate. Then, 100 µl of a solution of 15-lipoxygenase (200 U/ml) prepared in borate buffer (0.2 M, pH 9.0) was mixed with 25 µL of EO from C. nardus also prepared in borate buffer at different concentrations at 0.083 mg/ mL than at 2.2 mg/mL and incubated for 3 min at 25°C. A negative control without EO and a blank without enzyme were made. The reaction was initiated by the addition of 125 µL of a solution of linoleic acid substrate (234 µM) and the variation of the velocity was monitored at 234 nm for 3 min. The percentage of inhibition was calculated using the following formula: according to the formula: Radical scavenging capacity (%) = bsorbance Blank − Absorbance Sample Absorbance Blank 100 The concentrations were expressed in μg of extracts/μg of DPPH by the formula:

Concentration = Mass of DPPH
or Concentration = x Volume of EO Concentration of DPPH x Volume of DPPH calculated using the following formula: Where E is the activity of the enzyme without inhibitor (negative control), and S is the activity in the presence of the extract.

Flow cytometry analysis
LNCaP and HeLa cells were seeded at a concentration of 3×10 5 in 6-well plates and treated with either 220 µg/mL or 110 µg/mL of C. nardus EO for 72 h at 37°C. After the treatment, cells were harvested with trypsin, centrifuged and fixed with paraformaldehyde (4 %) for 15 min at room temperature, and then washed with PBS. 10 6 cells were prepared in suspension, centrifuged, and the supernatant removed. Then, 0.2 ml of FxCycle™ PI/RNase staining solution (Invitrogen, OR) was added to each tube and mixed well. Samples were incubated for 30 min at room temperature, protected from light, and analyzed by FACS using excitation at 488 nm; emissions were collected using a 585/42 bandpass filter.

Statistical analysis
In vitro experiments were performed in triplicate, each data point represents the average of at least three independent experiments. All data are presented as mean ± standard deviation. The data were analyzed by analysis of variance followed by the Turkey multiple comparison test. The analysis was performed using XLSTAT 7.1 software. A p value of < 0.05 was used as a criterion for statistical significance.
The EO of C. nardus exhibited an antioxidant activity of 102.19 ± 4.2 µg extract/µg DPPH compared to 0.11 ± 0.04 µg for gallic acid (p < 0.05) ( Table 2). The percentage of inhibition of DPPH radicals by the EO of C. nardus increased with concentration (Figure 3). In fact, at a concentration of 4.58 µg/mL of the C. nardus EO, a percentage of inhibition of DPPH radicals was 6.72% and 62.14% at 146.66 µg/mL (Figure 3). The antioxidant activity by the ABTS+• method, the EO has an activity of 0.009 ± 0.0004 µM TE/g against 2.66 ± 0.31 µM TE/g for the gallic acid ( Table 2). The results of inhibition of lipoxygenase by the EO are shown in Table  2. At a concentration of 0.083 mg/mL, the EO showed an inhibition of 0.5 ± 0.9% compared to 59.64 ± 2.12% of the gallic acid used as standard (p < 0.05). Moreover, for a concentration of 2.2 mg/mL of the EO of C. nardus, an inhibition of 25 ± 3% was obtained ( Table 2) The results of the C. nardus EO tested on LNCaP, HeLa, and P69 cells are shown in Table 3. An IC 50 was calculated from the dose-response curves (Figure 4) 58.0 ± 7.9 µg/mL, 142 ± 6 µg/mL and 100.9 ± 3.2 µg/mL (p < 0.05), respectively. Cisplatin presented IC 50 values of 4.4 ± 0.5, 7.8 ± 1.3, 11.4 ± 1.2 (p <0.05) on HeLa, LNCaP and P69 cells, respectively (Table 3). Figure 5 highlights the action of C. nardus EO on LNCaP prostate cancer cell morphology. Moreover, the cell cycle activity of the LNCaP cells was evaluated after C. nardus EO treatment ( Figure 6).
Antioxidant activity: Plants are generally a potential source of antioxidant molecules. Several studies have highlighted the strong antioxidant potential of EOs from medicinal plants used in Burkina Faso [14,15]. The EO from C. nardus has demonstrated antioxidant activity against both DPPH and ABTS+• cation radicals. The activity of gallic acid used as a standard is however greater. In addition, we identified antioxidant activity. Other studies showed that the EO from C. nardus has antioxidant activity [22], which could be explained by its high content of monoterpene alcohol (40.77%). Indeed, geraniol [40] is a significant monoterpene in the EO we studied. Compared to gallic acid used as standard, it should be noted that the activity of the EO from C. nardus is however, lower.
Anti-inflammatory capacity: Inhibition of lipoxygenase by the EO is concentration-dependent. At low concentration (0.083 mg/mL), there was virtually no inhibition (0.5 ± 0.9%), while at a higher concentration (2.2 mg/mL) a 25 ± 3% inhibition was noted. This antiinflammatory activity could be explained by its high citronellal content (33.06%). Previous studies have showed such activity by citronellal [41]. These results corroborate those of Kandimalla et al. [37]. In addition, several studies have shown anti-inflammatory activity of monoterpenes aldehydes; however the specificity of each compound within this group could contribute to increase or decrease activity [42]. Antiproliferative activity: Antiproliferative activity was studied to evaluate the putative antitumor properties of the EO from C. nardus on LNCaP and HeLa cells. Our results show for the first time that the EO inhibits the proliferation of both LNCaP and HeLa cancer cells. Antiproliferative activity was also discovered using noncancerous P69 epithelial cells. The antiproliferative activity of the EO from C. nardus was dependent on concentration for both cancer cells and non-cancer cells (Figure 4). Determination of the IC 50 showed that LNCaP cells are more sensitive than HeLa cells (58.0 ± 7.9 µg/mL  vs. 142 ± 6 µg/mL, respectively). The fact that the EO alters the cell proliferation is probably due to its high level of monoterpene alcohol (40.77%) of which geraniol (28.40%) and nerol (10.94%), as well to a significant amount of monoterpene aldehyde (33.32%) including citronellal (33.06%). The alcohol monoterpenes are known for their anticancer potential. Antiproliferative activities of citronellal [43] and geraniol [44,45] have already been reported. This could also explain the cytotoxicity observed using the P69 cells [46]. The most significant effect was observed in the LNCaP cells, we further analyzed the role of the EO from C. nardus on the cell morphology ( Figure 5) and cell cycle ( Figure 6) by cytometry.    Cisplatin, a compound used in cancer chemotherapy, was used as a reference compound, and exhibited superior activity to that of C. nardus EO on both HeLa and LNCaP cancer cells and on immortalized P69 prostate cells. Cisplatin is a pure compound, unlike EOs, which consist of a mixture of various molecules. However, the P69 cells are immortalized and this could, in part, justify the toxicity of the EO and cisplatin on these cells.
The cell cycle is closely linked to tumorigenesis. Irregular cell cycle regulation and the resultant uncontrolled cell growth that occurs as a consequence are common characteristics for most tumors [47]. Morphology of LNCaP cells were drastically modified, and this was due to the arrest of the cell cycle at the G2/M phase in a dosedependent manner.

Conclusion
C. nardus is commonly used in traditional medicine alone or in combination with other medicinal plants for the management of various diseases, mainly microbial and oxidative stress-related diseases in Burkina Faso, West Africa. Herein, we show that the EO of this plant has antioxidant properties through the inhibition of DPPH radicals and radical cation ABTS+•, anti-inflammatory inhibition of lipoxygenase and for the first time demonstrated anti-proliferative activity on various cell lines derived from prostate (LNCaP and P69) or cervix (HeLa). These data highlight the need for more in vitro research to identify the compound(s) responsible for these effects as well as the use of animal models to establish the pharmacokinetics and toxicity profiles of this EO, and to further quantify its putative anti-tumor effect.  of Burkina Faso for BB, Région Auvergne-Rhône Alpes, Fond Européen de Développement Régional (FEDER), Plan National de Recherche sur les Perturbateurs Endocriniens (13-MRES-PNRPE-1-CVS043), and Plan-Cancer 2016 for SB and JMAL. The funders had no role in the study design, data collection or analysis, decision to publish, or in the preparation of the manuscript.

Conflict of interest:
Authors state no conflict of interest