Phytochemical profiling and seasonal variation of essential oils of three Callistemon species cultivated in Egypt

The genus Callistemon comprises evergreen shrubs or small trees, widely cultivated as ornamentals and for essential oil production. Callistemon is well-recognized in folk medicine for its anti-cough, anti-bronchitis, and insecticidal activities. In the current study, we profiled the essential oil composition of the leaves of C. citrinus, C. rigidus and C. viminalis (Myrtaceae) collected during different seasons by GLC-MS coupled to multivariate data analysis. Antioxidant, anti-inflammatory and anti-proliferative activities of Callistemon essential oils were evaluated. A total of 29 compounds were tentatively identified. Oxygenated monoterpenes dominated in essential oils, where eucalyptol represented the major constituent in the three Callistemon species in all seasons. Multivariate data analysis including Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were applied to discriminate between different Callistemon species in each season and to investigate any correlation between the metabolic profile of each species within different seasons. As expected, PCA plot could discriminate the three Callistemon species in the four seasons. The dendrogram from HCA confirmed the results of PCA as it showed the same segregation pattern regarding the discrimination of different Callistemon species. C. viminalis showed more pronounced antioxidant activity than C. citrinus, exhibiting IC50 values of 1.40 mg/mL and 1.77 mg/mL, respectively. Meanwhile, C. rigidus showed very weak antioxidant activity. All oils showed membrane stabilization activity in hypotonic solution induced haemolysis assay, where C. viminalis showed potent membrane stabilizing activity exhibiting IC50 value of 25.6 μg/mL comparable to that of the standard drug, indomethacin (17.02 μg/mL). Nevertheless, Callistemon essential oils were not cytotoxic in HCT-116 and Hela human cancer cell lines.


Introduction
The genus Callistemon (Myrtaceae) consists of evergreen shrubs or small trees. It is widely used for essential oil production, and as ornamental, wind-breaking and degraded-land a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 flow rate of 1.41 mL/min. Mass spectra were recorded applying the following conditions: Ion source temperature, 200˚C; ionization voltage, 70 eV; (equipment current) filament emission current, 60 mA. Split mode injection of diluted samples (1% v/v) was applied with split ratio 1: 15.

Identification of essential oil components
Essential oil components were tentatively identified by comparison of their mass spectra and retention indices with those listed in NIST Mass Spectral Library, 2011; Wiley Registry of Mass Spectral Data 8 th edition; and data reported in literature [11][12][13].

Chemometric analysis
GLC/MS phytochemical profiling was subjected to chemometric analysis. Principal Component Analysis (PCA) comprises a first step in data analysis in order to provide an overview of all observations and samples to identify and evaluate groupings, trends and strong outliers [14,15]. Hierarchal Cluster Analysis (HCA) was then applied to allow clustering of different Callistemon species. The clustering patterns was constructed by applying the complete linkage method used for group building; this representation is more efficient when the distance between clusters is computed by Euclidean method [14,15]. For PCA and HCA, Unscrambler X 10.4 from CAMO (Computer Aided Modeling, AS, Norway) was employed.

Antioxidant activity
Antioxidant activity of Callistemon essential oils was investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay [16]. An aliquot (40 μL) of various concentrations of the essential oils (0.04-5.12 mg/mL) was added to 0.004% w/v DPPH in methanol (3 mL). Absorbance was recorded immediately against a blank using a UV-visible spectrophotometer (Milton Roy, Spectronic 1201). The decrease in absorbance at 515 nm was determined continuously, with the data being recorded at 1 min intervals until the absorbance was stabilized (16 min). Ascorbic acid was used as a reference compound. All experiments were performed in triplicates. The inhibition percentage (I%) was calculated according to the following equation: Where A blank = Absorbance of the blank (non-reduced DPPH) at t = 0 min and A sample = absorbance of the test sample at t = 16 min.

Anti-inflammatory activity
Membrane stabilization assay was used to assess the in vitro anti-inflammatory activity of Callistemon essential oils using hypotonic solution-induced erythrocyte hemolysis described by Shinde et al. [17].
Preparation of erythrocyte suspension. Whole blood was collected from rats via cardiac puncture under ether anesthesia into heparinized tubes. Blood was washed three times with 0.9% saline. The volume of saline was measured and reconstituted with isotonic buffer solution (pH 7.4) composed of 154 mM NaCl in 10 mM sodium phosphate buffer (pH 7.4) as 40% v/v suspension. The blood was then centrifuged at 3000 rpm for 10 minutes [17].
Hypotonic solution-induced hemolysis. Membrane stabilization activity of the essential oils was assessed using hypotonic solution-induced erythrocyte hemolysis [17]. Briefly, 0.5 mL of stock erythrocyte (RBCs) suspension was mixed with 5 mL of hypotonic NaCl solution (50 mM) in 10 mM sodium phosphate buffered saline (pH 7.4) containing the tested essential oil at a concentration of 7.81-1000 μg/mL. The control sample was composed of 0.5 mL of RBCs mixed with 5 mL hypotonic-buffered saline solution alone. Mixtures were incubated at room temperature for 10 min, then centrifuged at 3000 rpm for 10 min. Indomethacin was used as a reference standard. In 96 well plates, the absorbance (O.D.) of the supernatant was measured at 540 nm. The percentage inhibition of hemolysis or membrane stabilization percentage was calculated according to the method described by Shinde et al. [17]. The IC 50 value was defined as the concentration of the sample that inhibited 50% erythrocyte hemolysis under the assay conditions. %Inhibition of Hemolysis ðOr%Membrane Stabilization%Þ ¼ 100 x ðOD 1 À OD 2 =OD 1 Þ Where, OD 1 is the optical density of the hypotonic-buffered saline solution alone OD 2 is the optical density of the test sample in hypotonic solution. Anti-proliferative activity. The anti-proliferative activity of the essential oils was assessed against HCT-116 and Hela human cancer cell lines using MTT assay [13,18]. Exponentially growing cells were seeded at a density of 10×10 4 cells/well (HCT-116) and 15 x10 4 cells/well (Hela) in 96-well plates. Stock solutions of essential oils in dimethyl sulfoxide (DMSO) were prepared. Essential oils were subjected to two-fold serial dilutions in the respective media where the maximal concentration of DMSO did not exceed 1%. Doxorubicin was used as a positive control. Cells were treated with 100 μL of the tested essential oils at concentrations ranging from 0.002-1.0 mg/mL Cells were incubated for 24 h at 37˚C. Afterwards, 0.5 mg/mL of MTT was added, and the plates were incubated for additional 4 h. The formazan crystals produced by viable cells were dissolved in DMSO (100 μl) and subsequently shaken for 10 min at room temperature. The absorbance was measured at 570 nm using a Tecan Safire II (Crailsheim, Germany) spectrophotometric plate reader. The percentage cell viability was calculated using the following formula: % cell viability = (OD of treated cells / OD of control cells) x 100.

Data analysis
All experiments were carried out in triplicate. IC 50 value was determined as the concentration that resulted in 50% reduction in cell viability or inhibition of biological activity. IC 50 values were calculated using a four parameter logistic curve using SigmaPlot 14.0, SYSTAT Software (CA, USA). Data were presented as mean ± standard deviation.

GLC-MS analysis of the essential oils from different Callistemon species
Hydrodistillation of the fresh leaves of C. citrinus, C. rigidus, and C. viminalis yielded 0.43%, 0.84% and 0.41% w/w pale yellow essential oil, respectively. The identified components, their retention time, retention indices and percentages (average of three replicates for each species) for different seasons are summarized in Table 1. GLC/MS profiles of three Callistemon species collected during different seasons are displayed in supplementary material (S1-S4 Figs).
Twenty-nine components were tentatively identified in the three Callistemon species. The results in Table 1 demonstrate that oxygenated monoterpenes followed by monoterpenes are the major oil components of the three species accounting for (61.38% -94.42%) and (4.70% -30.37%) of the total identified components, respectively. Meanwhile, sesquiterpenes and other classes were present in low abundance.
However, essential oils from C. citrinus leaves from Western Himalayas revealed high content of α-pinene (32.3%) followed by limonene (13.1%) and α-terpineol (14.6%), whereas, 1,8-cineole was only 9.8% of the leaf oil which controverted with previous studies from other geographical regions [26]. Thus, remarkable qualitative and quantitative variations in essential oil composition could be traced among plants collected in different geographical regions and /or seasons which necessitates construction of a simple and efficient chemometric model that could discriminate closely related species collected in different seasons.

Discrimination of different Callistemon species by chemometric analyses
Different bar charts were constructed for the major identified components of Callistemon essential oils. As shown in Fig 1, bar charts exhibited quantitative and qualitative differences regarding the metabolic profile of each species in each studied season. There are very close correlations between different Callistemon species in different seasons, as all samples showed eucalyptol as the major metabolite. Metabolic profiling (29 components, Table 1) were subjected to both PCA and HCA to reveal the chemical variability, and the inter-relationships between the oils in each season and among different species.
PCA explained 99% and 100% of the variance of the data in spring and summer seasons, respectively, as shown in Fig 2A & 2B. The three species were significantly discriminated from each other, and each species was located in a different quadrant away from the other species. Loading plots showed that the main discriminating markers were eucalyptol, α-pinene and linalool. However, regarding autumn and winter season, PCA described about 100% of data discrepancy as presented in Fig 3A & 3B, where each species was completely segregated from each other. In addition to eucalyptol, α-pinene, and linalool, the loading plot showed that one main discriminating metabolic maker was α-terpineol, which highly influenced the segregation between the samples in winter season. However, for autumn both eucalyptol and α-terpineol were recognized as a marker for separation between different species.
Additionally, HCA was applied as unsupervised pattern recognition method in order to confirm results obtained by PCA. The dendrograms obtained for different seasons, displayed in Fig 4, revealed segregation of different Callistemon species into three main clusters endorsing the results of PCA. HCA dendrograms revealed the near distance of C. viminalis and C. rigidus in spring, summer and autumn as presented in Fig 4A, 4B & 4C, respectively. On the other side, regarding winter season, HCA showed nearness of C. citrinus, C. rigidus in relation to C. viminalis.
In an attempt to find the relationship between the phytochemical profile of each Callistemon species in different seasons, PCA was applied as shown in Fig 5A, 5B & 5C. Regarding C. citrinus and C. viminalis, PCA score plot demonstrated the discrepancy in the chemical composition of the essential oils collected in each season where they were completely segregated from each other with eucalyptol, α-pinene and α-terpineol as major metabolites with the highest impact on the separation of C. citrinus. In addition to eucalyptol, α-pinene, β-myrcene exhibited an influence on the segregation of C. viminalis in different seasons. For C. rigidus, a substantial difference was observed between essential oils constituents in spring and summer that are distanced from each other, with regard to that of autumn and winter that are closely related. From the loading plot, it was found that O-cymene and pseudolimonene were the main markers responsible for the segregation of C. rigidus in summer, however α-pinene discriminates the species in spring.
HCA results for C. citrinus, C. rigidus and C. viminalis in different seasons are illustrated in Fig 6A, 6B & 6C. The resulting dendrograms displayed the same pattern as all showed three main clusters. Regarding C. citrinus and C. viminalis, the dendrograms revealed the close distance between spring and summer, as they are grouped in the same cluster. On the contrary, C. rigidus, indicated a close association between winter and autumn.
The essential oil composition of all Callistemon species in different seasons exhibited common major constituents as eucalyptol, α-terpineol and α-pinene, which make their discrimination a major obstacle. By applying metabolomics fingerprinting in combination with chemometric analysis, such as PCA and HCA, this problem could be solved and was helpful to identify the plants as it does not only rely on major components, but takes into consideration all metabolic profiling [27].

Biological activity
Antioxidant activity. Essential oils have attracted attention for the plethora of bioactivities they possess. Callistemon essential oils were assessed for DPPH radical scavenging capacity. Antioxidant activity was presented herein as the concentration of essential oil that resulted Phytochemical profiling and seasonal variation of essential oils of three Callistemon species from Egypt in 50% free radical inhibition (IC 50 ). C. viminalis showed more pronounced antioxidant activity than C. citrinus, exhibiting IC50 values of 1.40 mg/mL and 1.77 mg/mL, respectively. Nevertheless, C. rigidus showed very weak antioxidant activity with IC 50 above the tested concentration range. Meanwhile, ascorbic acid exhibited IC 50 value of 14.2 μg/ml. Results were in agreement with previous studies. Essential oil from the leaves of C. citrinus showed free radical scavenging activity with IC 50 value of 4.02 mg/mL [28]. In another study, a pronounced free radical inhibitory activity (91.1 ± 0.3%) at a concentration of 1 mg/mL was observed for C. citrinus leaf essential oil, comparable to 0.1 mg/mL gallic acid (95.7 ± 2%) [29]. Phytochemical profiling and seasonal variation of essential oils of three Callistemon species from Egypt Anti-inflammatory activity. Inflammation is a normal defensive response to tissue injury or infection, functioning to combat invaders to remove damaged or dead host cells [30]. Erythrocytes membrane stabilization assay is considered a common tool to screen for anti-inflammatory candidates [17]. In this study, Callistemon essential oils showed inhibitory activity to the hemolysis of erythrocytes induced by hypotonic solution. C. viminalis showed potent membrane stabilizing activity exhibiting IC 50 value of 25.6 μg/mL. Results were comparable to Indomethacin (IC 50 17.02 μg/mL). Moreover, C. citrinus showed moderate activity with IC 50 value of 39.9 μg/mL. Meanwhile, C. rigidus displayed weak activity with IC 50 value of 217.1 μg/ mL.
The erythrocyte membrane is analogous to the lysosomal membrane. Thus, its stabilization serves as a parameter to assess the ability to stabilize the lysosomal membrane [17]. Stabilization of the lysosomal membrane is necessary to limit the inflammatory response through inhibition of the release of lysosomal constituents of activated neutrophils such as proteases and bactericidal enzymes. Exposure of erythrocytes to a hypotonic medium results in membrane lysis [31]. A possible mechanism for membrane stabilization activity of Callistemon essential oil observed herein could be attributed to the ability of essential oil constituents to integrate into cellular membranes, increasing the surface area to volume ratio of the cells that might be brought about by expansion of the membrane or shrinkage of the cell, as well as an interaction with membrane proteins [17]. Numerous terpenoids have been previously reported to possess anti-inflammatory activity. 1,8-cineole, was reported to inhibit the production of leukotrienes (LTB4) and PGE2 [32]. Furthermore, α-terpineol was reported to inhibit histamine release and reduce the production of inflammatory mediators [33]. Ahmed et al. reported that C. citrinus chloroform fraction exhibited membrane stabilizing anti-inflammatory potentials in Phytochemical profiling and seasonal variation of essential oils of three Callistemon species from Egypt hypotonic solution-induced hemolysis, results were comparable to acetylsalicylic acid, the standard drug [31].
Anti-proliferative Activity. The anti-proliferative activity of essential oils has been widely explored and numerous studies are now available in literature [10]. In this study, the anti-proliferative activity of Callistemon essential oils was assessed on HCT-116 and Hela human cancer cell lines using MTT assay. The three essential oils showed no cytotoxic activity. C. citrinus essential oil exhibited an IC 50 value of 0.60 mg/mL on HCT-116 cancer cell line, whereas, IC 50 values of 0.85 mg/mL and 0.51 mg/mL were recorded for C. rigidus and C. viminalis, respectively. Doxorubicin exhibited IC 50 value of 4.62 μM on the aforementioned cancer cell line. On the other hand, the IC 50 values recorded for C. citrinus, C. rigidus and C. viminalis on Hela human cancer cell line were 2.427, 3.428 and 1.928 mg/mL, respectively. These IC 50 values indicate that the essential oils are not cytotoxic.
Previous studies conducted by Kumar et al. showed that C. citrinus leaf essential oil was not cytotoxic to rat glioma (C-6), human colon cancer (Colo-205), human cervical cancer (SiHa) and human peripheral blood mononuclear cells (PBMCs) at concentrations up to 100 μg/mL [26]. In the same context, essential oils obtained from C. viminalis leaves were not cytotoxic to melanoma cells (HT144) at concentration (200 μg/mL) where only 40% reduction in percentage cell viability was observed [34].

Conclusion
The phytochemical profiling of the essential oils from three Callistemon species by GLC/MS showed that oxygenated monoterpenes represent the major class of the oil components with eucalyptol as the major secondary metabolite. The chemical profiles show high qualitative and quantitative similarities between species. Different chemometric analysis techniques were effectively applied as a discriminatory tool to differentiate between three Callistemon species, in each season, and within the same species in different seasons. C. viminalis essential oil exhibited pronounced membrane stabilization activity, which was equivalent to that of the standard drug, indomethacin. The three essential oils showed no cytotoxic activity against tested cancer cell lines. Future studies should be implemented to unravel other potential bioactivities of Callistemon essential oils.