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

  • Haidy A. Gad ,

    Contributed equally to this work with: Haidy A. Gad, Iriny M. Ayoub

    Roles Conceptualization, Data curation, Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

  • Iriny M. Ayoub ,

    Contributed equally to this work with: Haidy A. Gad, Iriny M. Ayoub

    Roles Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing

    Affiliation Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

  • Michael Wink

    Roles Conceptualization, Resources, Supervision, Writing – review & editing

    Affiliation Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, INF, Heidelberg, Germany

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

  • Haidy A. Gad, 
  • Iriny M. Ayoub, 
  • Michael Wink


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.


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 reclamation plants [1]. Callistemon originates from Australia, but is cultivated and introduced to most parts of the world [2]. Callistemon species possess attractive lanceolate leaves and characteristic 'bottlebrush' shaped flower spikes with prominent red stamens, thus, commonly named Bottlebrush [3]. The most widely cultivated member of this genus is C. citrinus, commonly known as Crimson or Lemon Bottlebrush [2]. C. viminalis is a small tree or shrub characterized by pendulous foliage and thus, was given the name Weeping Bottlebrush [2]. C. rigidus, commonly called Stiff or Erect Bottlebrush, is a stiff and upright shrub with red flower spikes [4].

Callistemon species produce essential oils. In folk medicine, Callistemon is well recognized for its insecticidal, as well as its anti-cough and anti-bronchitis activities [5]. The essential oils possess antimicrobial, antifungal, insecticidal, anthelmintic, antioxidant, anti-inflammatory and anti-nociceptive activities [58]. In China, Callistemon species, mainly C. viminalis is used in Traditional Chinese Medicine for treating hemorrhoids [9]. 1,8-Cineole figured as the predominant constituent reported in Callistemon essential oils. Other reported secondary metabolites include α-pinene, β-pinene, limonene, linalool, myrcene, and menthyl acetate [3]. The biological activity of an essential oil could not be ascribed to a single component but rather to the synergistic effects of the phytocomplex. This chemical complexity contributes to its biological activity, since each constituent of the phytocomplex is involved in the overall activity or may modulate the effects of the other constituents [10].

The main goal of this study is phytochemical profiling of essential oils from three Callistemon species (C. citrinus, C. rigidus and C. viminalis) in different seasons (spring, summer, autumn and winter) by GLC/MS. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied as pattern recognition techniques to discriminate between species and seasons. Moreover, essential oils were assessed for their antioxidant, anti-inflammatory and anti-proliferative activities.

Materials and methods

Plant material

Leaves of Callistemon citrinus, C. rigidus and C. viminalis (Myrtaceae) were collected in four different seasons from a private botanical garden in Giza, Egypt. Plants were kindly identified by Mrs. Trease Labib, Plant Taxonomy Consultant at the Ministry of Agriculture and former director of Orman Botanical Garden, Giza, Egypt. Voucher specimens were deposited in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University (PHG-P-CC; PHG-P-CR; and PHG-P-CV).

Isolation of the essential oil

The essential oils from fresh leaves (100 g) of Callistemon citrinus, C. rigidus and C. viminalis were obtained by hydro-distillation for 4 h using a Clevenger-type apparatus. The oil was collected, dried over anhydrous sodium sulfate, weighed and kept in sealed vials at 4°C for further analyses. The yield in % (w/w) was determined in triplicate, based on the initial plant weight.

GLC/MS analysis

Mass spectra were recorded using a Shimadzu GCMS-QP2010 (Kyoto, Japan) equipped with a split–splitless injector. Separation was achieved using an Rtx-5MS fused silica capillary column (30 m x 0.25 mm i.d. x 0.25 μm film thickness) (Restek, USA). The initial column temperature was kept at 45°C for 2 min and programmed to 300°C at a rate of 5°C/min, and kept constant at 300°C for 5 min. Injector temperature was 250°C. Helium was used as a carrier gas with a 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 8th edition; and data reported in literature [1113].

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 Ablank = Absorbance of the blank (non-reduced DPPH) at t = 0 min and Asample = 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 IC50 value was defined as the concentration of the sample that inhibited 50% erythrocyte hemolysis under the assay conditions.


OD1 is the optical density of the hypotonic-buffered saline solution alone

OD2 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 10×104 cells/well (HCT-116) and 15 x104 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. IC50 value was determined as the concentration that resulted in 50% reduction in cell viability or inhibition of biological activity. IC50 values were calculated using a four parameter logistic curve using SigmaPlot 14.0, SYSTAT Software (CA, USA). Data were presented as mean ± standard deviation.

Results and discussion

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 (S1S4 Figs).

Table 1. Chemical profile of the essential oils of Callistemon citrinus (CC), C. rigidus (CR) and C. viminalis (CV) in four different seasons.

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.

The major secondary metabolite of the three essential oils in different Callistemon species was eucalyptol (syn. 1,8-cineole) ranging from (71.27% - 81.70%), (69.15% -81.70%), (64.63% - 79.39%) and (55.69% - 80%) in spring, summer, autumn and winter, respectively, with the highest variation in summer and spring. Essential oils of the three Callistemon species in four seasons showed α-terpineol and α-pinene as major components in addition to eucalyptol. α-Thujene, β-pinene, O-cymene, ɣ-terpinene, terpinolene and 4-terpinenol were detected as common constituents in all Callistemon species.

Our results were consistent with previous reports on the essential oils obtained from the leaves of Callistemon species from different geographical regions. A study conducted on thirty Australian Callistemon species reported that 1,8-cineole was the major component (45–80%) of the majority of leaf essential oils. Other identified compounds include α-pinene (2–40%), limonene (2–9%) and α-terpineol (1–13%) [19]. Essential oil from the leaves of C. lanceolatus (syn. C. citrinus) from the north-eastern region of India exhibited 1,8 cineole (58.3%) as a major constituent followed by α-pinene, α-phellandrene, limonene and α-terpineol [20]. Likewise, the oil of C. citrinus from Reunion was found to be rich in 1,8-cineole (68.0%) followed by α-pinene and α-terpineol [21]. These results matched to a great extent the oil from the lower Himalayan region except for α-terpineol which was present at a lower percentage [22]. Essential oils from Brazil, were characterized by a high content of 1,8-cineole (77.0% and 65.0%) for C. citrinus and C. viminalis, respectively [23]. Essential oils from C. viminalis leaves were reported to possess 1,8-cineole (47.9%–82.0%) as the predominant constituent [24]. Similarly, oils of C. citrinus and C. rigidus from Cameroon were dominated by 1,8-cineole (73.8% and 79.1%, respectively) [25].

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.

Fig 1. Bar charts of the main identified components of Callistemon species in spring, summer, autumn and winter.

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.

Fig 2.

PCA score and loading plots of different Callistemon species (a) spring and (b) summer.

Fig 3.

PCA score and loading plots of different Callistemon species (a) autumn and (b) winter.

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.

Fig 4.

HCA of different Callistemon species (a) spring (b) summer (c) autumn (d) winter.

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.

Fig 5.

PCA score and loading plots of different Callistemon species (a) C. citrinus (b) C. rigidus (c) C. viminalis in all seasons.

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.

Fig 6.

HCA of different Callistemon species (a) C. citrinus (b) C. rigidus (c) C. viminalis in all seasons.

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 in 50% free radical inhibition (IC50). 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 IC50 above the tested concentration range. Meanwhile, ascorbic acid exhibited IC50 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 IC50 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].

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 IC50 value of 25.6 μg/mL. Results were comparable to Indomethacin (IC50 17.02 μg/mL). Moreover, C. citrinus showed moderate activity with IC50 value of 39.9 μg/mL. Meanwhile, C. rigidus displayed weak activity with IC50 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 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 IC50 value of 0.60 mg/mL on HCT-116 cancer cell line, whereas, IC50 values of 0.85 mg/mL and 0.51 mg/mL were recorded for C. rigidus and C. viminalis, respectively. Doxorubicin exhibited IC50 value of 4.62 μM on the aforementioned cancer cell line. On the other hand, the IC50 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 IC50 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].


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.

Supporting information

S1 Fig. GLC/MS chromatograms of the essential oils of C. citrinus (CC), C. viminalis (CV) and C. rigidus (CR) collected in spring.


S2 Fig. GLC/MS chromatograms of the essential oils of C. citrinus (CC), C. viminalis (CV) and C. rigidus (CR) collected in summer.


S3 Fig. GLC/MS chromatograms of the essential oils of C. citrinus (CC), C. viminalis (CV) and C. rigidus (CR) collected in autumn.


S4 Fig. GLC/MS chromatograms of the essential oils of a C. citrinus (CC), C. viminalis (CV) and C. rigidus (CR) collected in winter.



Iriny M. Ayoub is thankful to the Science and Technology Development Fund in Egypt (STDF, project ID 25448) for funding the postdoctoral fellowship in Germany, where she performed the anti-proliferative activity against HCT116 and Hela cancer cell lines.


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