Received: 10 February 2021 | Revised: 25 March 2021 | Accepted: 29 April 2021

DOI: 10.1111/jfbc.13767

ORIGINAL ARTICLE

Chemical composition of Callistemon citrinus (Curtis) Skeels

aerial part essential oil and its pharmacological applications,
neurodegenerative inhibitory, and genotoxic efficiencies

Roktim Gogoi1,2 | Twahira Begum1,2 | Neelav Sarma1,2 | Sudin Kumar Pandey1,2 |

Mohan Lal1

1
Medicinal Aromatic and Economic Plants
Group, Biological Science and Technology
Division, CSIR-­North East Institute of
Science and Technology (NEIST), Jorhat,
Assam, India
2
AcSIR-­Academy of Scientific and Innovative
Research, Ghaziabad, Uttar Pradesh, India
Correspondence
Mohan Lal, Medicinal Aromatic and
Economic Plants Group, Biological Science
and Technology Division CSIR-­North East
Institute of Science and Technology (NEIST),
Jorhat, Assam 785006, India.
Email: drmohanlal80@gmail.com
Funding information
CSIR-­New Delhi, India, Grant/Award
Number: OLP-­2034 and HCP-­0 07
Abstract
Callistemon citrinus aerial part essential oil (CCEO) was analyzed for chemical com-
positions using GC/MS. Pharmacological activities such as neurodegenerative in-
hibitory, antioxidant, anti-­
inflammatory, antimicrobial, and acetylcholinesterase
activities were evaluated using the standard methodologies. Genotoxicity was in-
vestigated using Allium cepa assay. GC/MS analysis identified 27 compounds; euca-
lyptol (55.40%) was the major component. Radical scavenging activity showed IC50
value of 16.71 μg/mL. Protein denaturation assay showed IC50 value of 21.19 μg/
mL and 19.53μg/mL in protease inhibitor activity. MIC assay revealed antimicrobial
potential of CCEO against microbial strains B. cereus at 2.00 mg/mL, S. typhimurium
at 4.50 mg/mL, S. mutans at 2.50 mg/mL, C. albicans at 4.00 mg/mL, and S. cerevisiae
at 4.50 mg/mL concentrations. Mitotic index value (MI) of CCEO showed negligible
genotoxicity with MI 17.25% close to distilled water 18.22%. Acetylcholinesterase
strong inhibitory activity of CCEO was observed from IC50 = 6.335 μg/mL. CCEO
could be a cheap and easy source for the extraction of the pure compound eucalyptol
and possess various biological activities which increase its pharmacological value as
well as its applicability in the field of flavor and fragrance industries.
Practical applications
Eucalyptol is the major component of many pharmaceutical and food/flavor indus-
tries. The present investigation provides a new source for the isolation of pure com-
pound eucalyptol in cost-­effective way. Additionally, the essential oil could also be
used for the pharmaceutical formulation of antioxidant, inflammation inhibitory, and
neurodegenerative inhibitory drug preparation under the safety range concentration.

KEYWORDS

anti-­inflammatory, antimicrobial, antioxidant, Callistemon citrinus, essential oil, genotoxicity

1 | I NTRO D U C TI O N utilized for the treatment of many diseases (Manikandan et al., 2020).
Plants used in conventional medicine possess large number of bio-
Plants are considered as an important source of medicine since thou- active compounds necessary for the treatment of chronic as well as
sands of years. In all the continents back to prehistory, plants were infectious diseases (Leelavathi et al., 2010). Plants synthesize a wide

J Food Biochem. 2021;00:e13767. wileyonlinelibrary.com/journal/jfbc © 2021 Wiley Periodicals LLC. | 1 of 12

https://doi.org/10.1111/jfbc.13767
2 of 12 | GOGOI et al.
range of chemical compounds also known as primary and secondary
metabolites, which can be categorized by their chemical class and
biosynthetic origin (Manikandam et al., 2017). Among those second-
ary metabolites, essential oils are used as preservatives and medi-
cines since ancient times along with their ability to enhance flavor
and aroma to food (Edris, 2007).
Callistemon citrinus (Curtis) Skeels, one of such essential oil-­bearing
plants commonly known as “bottlebrush” is widely cultivated around
the world (Wrigley & Fagg, 2007). The plant belongs to Myrtaceae
family possesses lanceolate leaves with its characteristic flower in the
shape of “bottlebrush” resembled by the flower spikes with prominent
red or crimson red stamen (Oyedeji et al., 2009). C. citrinus is principally
distributed in Australia, South America, and tropical Asia which played
an important role in traditional aboriginal medicines. Some of the spe-
cies of Callistemon were used by the aboriginal peoples of Australia ex-
tensively for different traditional uses (Williams, 2011). The Callistemon
species have been well recognized traditionally and used for anti-­
bronchitis, anti-­cough, and insecticidal activities (Goyal et al., 2012).
They are also widely used for essential oil production, degraded-­land
reclamation, forestry, weed controller, bioindicators for environmental
management, and ornamental horticulture (Opeoluwa et al., 2009). An
article by Netala et al. (2015) reported that different parts of C. citrinus
are used by rural people of India; moreover aerial parts of the plant are
used traditionally in ethnic tribal communities. Related species such as
C. viminalis, C. citrinus, and C. lanceolatus from India, Australia, Egypt,
Reunion Island, and Pakistan have been reported earlier for their es-
sential oil compositions. Eucalyptol also known as 1, 8-­cineole was
the predominant constituent of the essential oils (Jamzad et al., 2014).
Moreover, the essential oil of this genus is known to possess anti-
microbial, antihelmintic, antifungal, anti-­
inflammatory, antioxidant,
antithrombotic, anti-­
staphylococcal, and anti-­
nociceptive proper-
ties (Kamal & Fareeda, 2017; Kumar et al., 2011; Zubair et al., 2013).
Among the other species, C. citrinus has a great medicinal value with
pollution tolerant ability. Phytochemical analysis dealing mostly with
leaf and flower extracts revealed the species to be rich with bioactive
compounds such as different polyphenols (phenolic acids and flavonol
glycosides); terpenes (Radulovic et al., 2015).
In this context, it can be said that in a biological system reac-
tive oxygen species (ROS) acts as a free radical giving rise to oxida-
tive stress (Gutteridge & halliwell, 1994; Maxwell, 1995; Sies, 1997)
which is associated with a variety of ailments such as AIDS, athero-
sclerosis, cancer, diabetes mellitus, hypertension, and inflammation
(Halliwell & Gutteridge, 1989). Thus the potent natural source of
anti-­oxidant is required to combat the harmful damaging activity of
the free radicals on the human body. Similarly, inflammation tends
to be a defensive response which is normally induced due to some
endogenous or exogenous stimuli causing infection or injury to the
host cells (Wei et al., 2015). Long-­term and severe inflammatory re-
sponses can be linked with many diseases such as bacterial infec-
tion, autoimmune diseases, arthritis, viral infection, atherosclerosis,
periodontitis, and chronic hepatitis (James et al., 2005). Therefore,
search and formulation of new natural anti-­inflammatory agents
would be beneficial for the treatment of many chronic ailments
originating from inflammatory responses. Nowadays, there are many
classes of antimicrobial drugs prescribed in conventional medicines
but these agents are showing reduced efficacy due to the adap-
tive drug-­resistant ability of microbial strains (Hoseini et al., 2014,
2015; Rad et al., 2014). As mentioned, emergence of such resistant
microbial strains affect the success rate of conventional therapies
seriously and thus there is a need to search for a new alternative of
antimicrobial agents (Miri et al., 2013; Rad et al., 2014). Therefore,
the present investigation was tried to the search for new natural or-
igin antimicrobial agent. One of the most important and demanding
search for drugs is related to central nervous system-­related dis-
eases. Cognitive disorders have been treated by the use of essential
oils for many years on an empirical base which was proved scientif-
ically (Perry et al., 2003; Savelev et al., 2003). Two percent popula-
tion of the developed countries are affected by Alzheimer's disease
(AD), a neurodegenerative disorder (Mattson, 2004) and the drugs
like donepezil, galantamine, rivastigmine, and tacrine approved by
FDA has severe side effects though they decrease the development
of the disease (Khalid et al., 2004), therefore, search for alternative
anti-­acetylcholinesterase compounds with less negative effect and
efficient ability is almost necessary. Regardless the wide applica-
tions of essential oils, its acute toxicity studies have not been well
explored. Therefore, prompted by the lack of detailed chemical data,
its known ethnopharmacological use the present study investigates
the possible biological properties of C. citrinus aerial part essential
oil (CCEO) through antioxidant, anti-­inflammatory, antimicrobial,
anti-­cholinesterase activities as well as genotoxicity with the wish to
establish a link between human consumption and beneficial effects
of the species.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Chemicals used in the study
In the present experiment methanol (CH3OH) HPLC grade, ferric-­
chloride (FeCl3), sodium-­
carbonate (Na2CO3), DPPH that is,
2,2-­diphenyl1picrylhydrazyl (C18H12N5O6), aluminum chloride
(AlCl3), potassium ferricyanide (K3Fe(CN)6), Tris buffer (C4H11NO3),
ethyl methanesulfonate (EMS) (C3H8O3S), acetocarmine, Mueller–­
Hinton agar (MHA), and potato dextrose agar (PDA) were procured
from HiMedia (India); while trichloroacetic acid (C2HCl3O2), acetyl-
cholinesterase (AChE), galanthamine hydrobromide, acetylcholine
chloride, and acetylthiocholine iodide were purchased from Sigma-­
Aldrich (Germany). The other requirements like diclofenac sodium
(100 mg) and fresh eggs were purchased from Jorhat local market
(Assam).
2.2 | Plant sample and essential oil extraction
Callistemon citrinus aerial parts were collected from the experimen-
tal farm of CSIR-­NEIST, Jorhat, India with GPS location of latitude:
GOGOI et al. | 3 of 12

26.7378 °N longitude: 94.1570 °E. The identification of the mate-
rial was performed by Dr. Mohan Lal (Senior Scientist, MAEP group)
and a specimen herbarium was deposited in departmental herbar-
ium of CSIR-­NEIST, Jorhat vide deposit number RRLCC-­017. For the
extraction of essential oil, 300 gm of shade-­dried aerial parts were
transferred to three liters capacity Clevenger apparatus to which
distilled water was added. The temperature of the apparatus was set
at 60°C for three and half hours then the distilled oil was collected
in a glass vial. Anhydrous sodium sulfate (Na2SO 4) was treated to the
extracted essential oil to remove the excess moisture content, and
further stored at 4°C in a sealed tube, until used for chemical and
biological analysis (Gogoi et al., 2020).
spectrophotometer). The absorbance was read at 517 nm using the
standard protocol suggested by (Blois, 1958). C. citrinus aerial part
essential oil with varying concentrations ranging from 5 to 30 µg/mL
was prepared in methanol to which 0.2 mM DPPH solution (1 mL)
was added. The reaction mixture was then incubated in dark at 37°C
for 30 min and was measured with UV/Vis spectrophotometer at
517 nm absorbance. The percentage scavenging effect was calcu-
lated according to the following formula
[( ) ]
DPPH scavenging effect ( % ) = Acontrol − Asample ∕Acontrol × 100 .
where Acontrol = absorbance of DPPH and Asample = absorbance of
DPPH + essential oil sample/standard of various concentrations.

2.3 | Chemical analysis of C. citrinus essential oils

through GC/MS 2.5 | Reducing power assay

The chemical analysis of Callistemon citrinus (aerial part) essential Different concentrations of C. citrinus essential oil (5–­30 µg/mL)
oil was performed using GC/MS which was programmed as shown were prepared in 0.2 M phosphate buffer (pH 6.6) and 1.1% potas-
below: sium ferricyanide (K3Fe(CN)6) (1.25 mL) to determine the reducing
power using the standard protocol (Oyaizu, 1986). The reaction mix-
GC/MS ture was then incubated for 20 min at 50°C. After that 10% acetic

Instrument Agilent Technologies Gas acid (1.25mL) was added and centrifuged at 1,008.06 g. An equal
Chromatograph coupled with mass amount of distilled water was added to the supernatant collected
selective detector MSD 5975C (1.5 mL) and 0.1% FeCl3 (0.3 mL) was added. Genesis 10 UV spectro-
Column HP-­5MS column (30 m × 0.25 mm i.d.) photometer was used to check the absorbance of the sample mix-
Film thickness 0.25 µm tures at 700 nm against a blank.
Oven temperature 40°C for 2 min
250°C at 5°C/min
300°C at 30°C/min for 10 min 2.6 | In vitro anti-­inflammatory and protein
Injector temperature 250°C denaturation assay
Sample injection volume 1 μL
Split ratio 1:20 Callistemon citrinus aerial part essential oil was evaluated for
Carrier gas 2+
Helium (He ) (1 mL/min) anti-­
inflammatory activity using the standard protocol (Sangita
Ionization voltage 70 eV et al., 2012). Five milliliters of the reaction mixture was prepared
Flow rate 1 mL/min which consists of egg albumin from fresh hen's egg (0.2 mL ),

Scanning range 45–­650 amu

Mass spectra value of the components was matched with Willey
mass spectral/NIST library and compared with Kovat's index on
HP-­5MS column (Adams, 2017; Goodner, 2008; Kovats, 1965). The
external standard method was used for quantification in which the
calibration curves were generated by running the standard com-
pounds in Gas chromatography in the same conditions as that of the
essential oil. Kovat's method was used to calculate the RI (retention
indices) value of the identified compounds using alkenes (C8–­C32) as
a standard.
2.4 | Antioxidant assay
phosphate-­buffered saline with pH 6.4 (2.8 mL), and essential oil/
drug (2 mL) of different concentrations (50–­300 µg/mL). The double
distilled water of equal volume was taken as a control. The reaction
mixtures were incubated in a BOD incubator for 15 min at 37 ± 2°C
and were then heated in the hot water bath for 5 min at 70°C. The
mixture was allowed to cool before absorbance was read at 660 nm.
The vehicle was used as a blank. Reference drug diclofenac sodium
was used at the concentrations of 5, 10, 15, 20, 25, and 30 µg/mL
for the absorbance determination. All experiments were performed
three times and the average values were taken as final values. The
inhibition percentage of protein denaturation was calculated using
the following formula
[ ]
% inhibition = 100 × Vt ∕Vc − 1 .

The antioxidant assay was performed using DPPH free radical where Vc is the absorbance of the control and Vt is the absorbance for
scavenging assay by spectrophotometric method (Genesis 10 UV the test sample.
4 of 12 | GOGOI et al.
2.7 | Protease inhibitory activities
Protease inhibitory activity was analyzed using the standard method
by (Kunitz, 1947). CCEO was prepared in the concentrations of
5–­30 µg/mL with 7.5 mL of reaction mixture containing equal vol-
ume of 1 mol/L trypsin solution (1 mL) and various concentration of
CCEO sample (1 mL). They were incubated at 37°C for 5 min. After
that 0.1% casein (2 mL) solution was added, mixed properly, and in-
cubated for 15 min. The reaction was stopped by adding 60% per-
chloric acid (2.5 mL) to the reaction mixture. The absorbance was
read at 280 nm (Genesis 10 UV spectrophotometer). Protease inhibi-
tory activity was measured using the formula
[ ]
Protease inhibitory % = Vcontrol − Vtest ∕Vcontrol × 100
where Vcontrol was the absorbance of the control and Vtest was the ab-
sorbance of the CCEO sample.
2.8 | Antimicrobial activities
2.8.1 | Microbial strains used for
antimicrobial screening
Four bacterial strains were taken for antimicrobial screening out of
which Bacillus subtilis (ATCC-­11774), Bacillus cereus (ATCC-­10876),
and Staphylococcus aureus (ATCC-­11632) are the gram-­positive
bacterial strains; Salmonella typhimurium (ATCC-­13311) is the gram-­
negative bacteria. Five fungal strains namely; Aspergillus fumigatus
(ATCC-­204305), Aspergillus niger (ATCC-­16885), Saccharomyces
cerevisiae (ATCC-­9763), Candida albicans (ATCC-­66027), and
Streptococcus mutans (ATCC-­25175). Fluconazole (10 µg/disk) and
ciprofloxacin (10 µg/disk) were used as standard drugs for antifungal
and antibacterial activities, respectively.
2.8.2 | Disk diffusion and minimum inhibitory
concentration (MIC) assays
The disk diffusion method was carried out using MHA for bacteria
and PDA for fungus. The antibacterial and antifungal activities of
Callistemon citrinus aerial part essential oil were determined in the
concentrations of 50, 100, 250, and 500 μg/mL. Inoculants for bac-
teria and fungus were prepared in Mueller–­Hinton broth (MHB) at
37°C for 16–­18 hr and potato dextrose broth (PDB) at 28°C for 48 hr,
respectively, whose optical density was adjusted to 0.5 Mc Farland
standard. Minimum inhibitory concentration of CCEO against bacte-
ria was determined using broth micro-­dilution method in microtiter
plates consisting of 96 wells. The essential oil, as well as standards
and the micro-­organisms used were poured in 1:1 ratio that is, 100
μL of microbial strains suspended in MHB were added to the same
volume of different concentrations of CCEO/standard which was
then incubated at 37°C for 24 hr. Ten microliters of 2,3,5 triphenyl
tetrazolium chloride (TTC) was added to the suspension and incu-
bated for 30 min. Color conversion from transparent to red distinctly
indicated the presence of bacterial growth (Rafael et al., 2009).
2.9 | Acetylcholinesterase activity assay
Acetylcholinesterase activity of CCEO was analyzed using the
standard methodology (Ellman et al., 1961). Acetylcholinesterase
(AChE) with the concentration of 0.04 U/mL and 75 mM acetylthi-
ocholine iodide (ATCI) were dissolved in phosphate buffer (0.1 M)
with pH 8.0. 5,5'-­dithiobis-­(2-­nitrobenzoic acid) (DTNB 0.01 M) was
prepared in 0.1 M phosphate buffer (10 mL) having pH 7.0 consisting
15 mg of sodium bicarbonate (NaHCO3). Assessment of AChE inhibi-
tion was assessed by the spectrophotometric method of Ellman's.
Fifty microliters of inhibitor solution (essential oil sample/standard
cholinesterase inhibitor drug) and 0.5 mL of AChE were mixed in a
test tube and was set at 25 ºC in an incubator. DTNB solution (100
µL) and 2.4 mL buffer with pH 7.0 were added to the test tube and in-
cubated at 25°C for 5 min. The reaction was again started by adding
40 µL of ATCI and again were incubated at 25 ºC for another 20 min.
After that the absorbance of the reaction mixtures was measured at
412 nm under UV/Vis spectrophotometer. Methanol was taken as
reaction blank, methanol in place of standard or sample with other
reagents was considered as control. The percentage of inhibition to-
ward cholinesterase activity was calculated as follows:
Inhibition activity (%)
= (Absorbance of control − Absorbance of sample∕Absorbance of Control) × 100
2.10 | Test for genotoxicity (Allium cepa assay)
Allium cepa (2n=16) assay was performed to check the genotoxic-
ity of CCEO which was procured from Jorhat vegetable market. The
outer dried scales were carefully peeled after washing the onion
bulbs properly. The protocol for present bioassay was according to
(Babatunde & Bakare, 2006; Rank & Nielsen, 1993). C. citrinus aerial
part EO of 1 µg/mL of concentrations was considered to check the in-
hibition of root growth. Distilled water was taken as negative control
and ethyl methanesulfonate (EMS) as positive mutative agent. The
experiment was carried out using three onion bulbs for each con-
centration of the essential oil sample. The bases of the bulbs were
suspended for 72 hr on the surface of the essential oil, positive and
negative control in 100 mL beakers which were changed regularly.
After that, root length emerged from onion bulbs was measured in
cm using a ruler for each concentration after the completion of the
exposure time. The percentage inhibitions of root growth were cal-
culated for all the samples including the positive and negative con-
trol (Fiskesjo, 1985). Genotoxic effects were further confirmed by
evaluating the chromosomal aberration initiation by the fixation of
bulb root tips in 3:1 ethanol: glacial acetic acid (v/v) and were hydro-
lyzed in 1 N HCL for 5 min at 60°C and washed in distilled water.
Induction of chromosomal aberration was checked by preparing
GOGOI et al.
five slides containing root tips each from different essential oil con-
centrations and control which was stained using acetocarmine. The
slides were then observed at a magnification of 1000X. The mitotic
index and aberrant cell frequency (%) were calculated according to
the formula suggested by (Bakare et al., 2000; Fiskesjo, 1997).
Number of dividing cells
Mitotic index =
500 observed cells
Aberrant cell frequency (%)
Numbers of aberrant cells
= × 100
Total cells scored at each concentration of essential oils
3 | R E S U LT S
3.1 | GC/MS analysis of CCEO
C. citrinus aerial parts yielded a total of 0.94% of essential oil (v/w)
in Clevenger apparatus. Gas chromatography–­mass spectroscopy
analysis detected 27 compounds with identified area percentage
of 95.55%. The major compound detected was eucalyptol (55.40%),
other compounds detected with good area percentage were
3-­carene (16.63%), α-­terpineol (5.98%), limonene (4.73%), β-­cymene
(4.28%), and α-­phellandrene (2.26%) (Figure 1). GC/MS analysis of
CCEO revealed monoterpene hydrocarbons (24.87%), oxygenated
monoterpenes (63.16%), sesquiterpenes hydrocarbons (01.77%),
oxygenated sesquiterpenes (00.92%), esters (00.07%), alkylbenzene
(04.28%), and others (00.48%) (Table 1).
3.2 | Antioxidant activities
In DPPH free radical scavenging assay, ascorbic acid and CCEO
showed antioxidant activity with IC50 value of 17.45 μg/mL and
16.71 μg/mL, respectively (Table 2). Reducing power activity
F I G U R E 1 Major compounds present in Callistemon citrinus aerial part essential oil (a. Eucalyptol, b. 3-­C arene, c. α-­Terpineol, d.
Limonene, and e. β-­Cymene)
| 5 of 12
showed stronger antioxidant potential of CCEO with an absorbance
of 0.492 ± 0.026 AU, better than the ascorbic acid with an absorb-
ance of 0.451 ± 0.002 AU (Table 3).
3.3 | Anti-­inflammatory activities
Anti-­inflammatory albumin denaturation assay showed inhibition
of 71.66 ± 0.024% by CCEO, whereas sodium diclofenac showed
59.11 ± 0.028% at 30 μg/mL concentration. The IC50 value of CCEO
was 21.19 µg/mL and 26.35 µg/mL for sodium diclofenac (Table 4).
Protease inhibitor assay showed IC50 = 19.53 µg/mL by CCEO and
IC50 = 28.42 µg/mL by sodium diclofenac (Table 5).
3.4 | Anti-­microbial activity (disk diffusion)-­dda and
minimum inhibitory concentration-­MIC assay
The dda revealed that CCEO possess highest inhibition zones
against bacterial strains B. cereus 20.67 ± 0.002 mm, S. typhimurium
11.00 ± 1.00 mm, and S. mutans 17.00 ± 0.012 mm and fungal strains
C. albicans 17.68 ± 1.53 mm and S. cerevisiae 12.67 ± 1.53 mm, re-
spectively. But there were no inhibitory effects of C. citrinus against
bacterial strains B. subtilis and S. aureus and fungal strains A. fumiga-
tus and A. niger (Table 6). Minimum inhibitory concentration assay
(MIC), again revealed antimicrobial potential of CCEO against bacte-
rial strains B. cereus at 2.00 mg/mL, S. typhimurium at 4.50 mg/mL,
and S. mutans at 2.50 mg/mL, similarly against the fungal strains C.
albicans at 4.00 mg/mL and S. cerevisiae at 4.50 mg/mL concentra-
tions. Standard antibacterial drug ciprofloxacin showed inhibition
zones of 19.00 ± 0.002 mm for B. cereus which was less than CCEO
and antifungal drug fluconazole showed 10.00 ± 0.046 mm and
2.50 ± 0.004 mm against C. albicans and S. cerevisiae, respectively
(Table 7).
6 of 12 | GOGOI et al.

TA B L E 1 Gas chromatography/mass
Name of the
a b spectroscopy profiling of Callistemon
Sl. No compound RT Area % KI KI
citrinus aerial part essential oil
1 α-­Thujene 6.95 1.25 923 924
2 α-­Phellandrene 7.19 2.26 1,001 1,003
3 3-­C arene 9.70 16.63 1,008 1,009
4 Isoamyl butyrate 10.04 0.07 1,010 1,010
5 α-­Terpinene 10.19 0.11 1,014 1,018
6 Limonene 10.51 4.73 1,029 1,029
7 β-­Cymene 10.52 4.28 1,030 1,031
8 Eucalyptol 10.87 55.40 1,033 1,035
9 γ-­Terpinene 11.94 0.56 1,061 1,062
10 Isoterpinolene 13.21 0.30 1,085 1,088
11 L-­Pinocarveol 13.72 0.15 1,141 1,145
12 Trans-­sabinol 14.29 trace 1,136 1,139
13 Terpinen-­4-­ol 15.41 1.22 1,177 1,178
14 α-­Terpineol 15.62 5.98 1,190 1,191
15 Linalyl formate 17.15 0.48 1,192 1,192
16 Fenchol 17.77 0.07 1,112 1,115
17 Trans-­C arveol 19.03 trace 1,201 1,217
18 Cis-­Geraniol 20.64 0.22 1,228 1,229
19 Thymol 22.72 0.12 1,292 1,297
20 Eugenol 25.16 trace 1,358 1,359
21 Methyleugenol 27.15 trace 1,401 1,415
22 Caryophyllene 27.75 0.31 1,417 1,418
23 Alloaromadendrene 28.55 0.25 1,453 1,458
24 Viridiflorene 30.85 0.14 1,495 1,497
25 δ-­C adinene 31.96 0.10 1,518 1,519
26 Spatulenol 34.07 0.63 1,576 1,578
27 Guaiol 34.70 0.29 1,597 1,599
Total area = 100% IC = 95.55% UIC = 4.45%
Monoterpene hydrocarbons Sl No. 1,2,3,6 24.87%
Oxygenated monoterpenes Sl No. 63.16%
8,11,12,13,14,16,17,18
Sesquiterpenes hydrocarbons Sl No. 5,9,10,22,23,24,25 01.77%
Oxygenated sesquiterpenes Sl No. 21,26,27 00.92%
Esters Sl No. 4 00.07%
Alkylbenzene Sl No. 7,20 04.28%
Others Sl No. 15 00.48%
a
KI = KI Literature (Goodner, 2008; Kovats, 1965; Adams, 2017).
b
KI = KI Reported, Sl No. = Serial number, % = Percentage, IC = Identified compounds,
UIC = Unidentified Compounds.

3.5 | Acetylcholinesterase inhibitory activity 3.6 | Genotoxicity Allium cepa assay

As we know that neurodegenerative disease can be treated by inhib-
iting AChE enzyme. Therefore, CCEO was tested for AChEI activity
and was compared with galanthamine. CCEO showed IC50 value of
6.335 µg/mL; which was a little lower than galanthamine IC50 value
of 6.652 µg/mL (Table 8).
Allium cepa assay of CCEO-­t reated root showed 0.70 cm growth,
distilled water 1.39 cm, while ethyl methanesulfonate (EMS)-­
treated onion bulb root growth showed (0.26 cm) comparatively
very retarded growth (Table 9). Mitotic index value of CCEO-­
treated onion bulbs again showed negligible genotoxicity with
GOGOI et al. | 7 of 12

TA B L E 2 DPPH free radical scavenging

Sample/standard (% of inhibition ± SD) for ascorbic (% of inhibition ± SD) for
activities of Callistemon citrinus aerial part
concentrations (μg/mL) acid IC50 = 17.45 μg/mL CCEO IC50 = 16.71 μg/mL
essential oil
5 20.11 ± 0.012 18.67 ± 0.004
10 38.67 ± 0.003 32.05 ± 0.021
15 47.21 ± 0.012 52.17 ± 0.001
20 59.65 ± 0.011 61.28 ± 0.004
25 63.01 ± 0.024 68.92 ± 0.012
30 75.87 ± 0.018 78.25 ± 0.042

Abbreviations: %, percentage; CCEO, Callistemon citrinus aerial part essential oil; IC50, 50%
inhibition; SD, standard deviation.

TA B L E 3 Reducing power activity of

Sample/standard concentrations Ascorbic acid (absorbance in CCEO (absorbance
Callistemon citrinus aerial part essential oil
(μg/mL) AU ± SD) in AU ± SD)

5 0.078 ± 0.002 0.095 ± 0.014

10 0.151 ± 0.012 0.178 ± 0.004
15 0.238 ± 0.002 0.224 ± 0.026
20 0.305 ± 0.086 0.317 ± 0.001
25 0.383 ± 0.001 0.408 ± 0.016
30 0.451 ± 0.002 0.492 ± 0.026

Abbreviations: AU, absorbance unit; CCEO, Callistemon citrinus aerial part essential oil; SD, standard
deviation.

TA B L E 4 Anti-­inflammatory activity of
Sample/standard % of inhibition for sodium % of inhibition for CCEO
Callistemon citrinus aerial part essential oil
concentrations (μg/mL) diclofenac IC50 = 26.35 μg/mL IC50 = 21.19 μg/mL

5 06.35 ± 0.006 13.56 ± 0.028

10 12.12 ± 0.014 19.22 ± 0.004
15 21.05 ± 0.004 34.50 ± 0.002
20 35.78 ± 0.021 48.15 ± 0.001
25 48.20 ± 0.001 59.25 ± 0.001
30 59.11 ± 0.028 71.66 ± 0.024

Abbreviations: CCEO, Callistemon citrinus aerial part essential oil; IC50, 50% inhibition.

TA B L E 5 Protease inhibitory activity of Callistemon citrinus aerial part essential oil

Sample/standard concentration % of inhibition for sodium % of inhibition for IC50 value for sodium IC50 value for
(μg/mL) diclofenac CCEO diclofenac (μg/mL) CCEO (μg/mL)

5 12.42 ± 0.021 19.27 ± 0.008 IC50 = 28.42 IC50 = 19.53

10 18.04 ± 0.016 25.98 ± 0.001
15 25.32 ± 0.082 38.07 ± 0.004
20 32.46 ± 0.012 50.15 ± 0.001
25 42.66 ± 0.014 63.11 ± 0.002
30 56.45 ± 0.022 75.32 ± 0.014

Abbreviations: %, percentage; CCEO, Callistemon citrinus aerial part essential oil; IC50, 50% inhibition.

MI (mitotic index) 17.25% which was close to distilled water MI CCEO, and 18.40% for EMS (Table 11; Figure 2). Negative geno-
18.22% but comparatively very far from EMS MI 4.96% (Table 10). toxicity result of CCEO may be due to the presence of eucalyptol
Chromosomal aberration test revealed safe properties of CCEO, as the major constituent of CCEO comprising 55.40% of its total
where a total aberration of 2.00% for distilled water, 4.00% for area percentage.
8 of 12 | GOGOI et al.

TA B L E 6 Antimicrobial activities of Callistemon citrinus aerial part essential oil

Zone of inhibition (mm ± SD)

Ciprofloxacin Fluconazole
Microbial strains 50 μg/mL 100 μg/mL 250 μg/mL 500 μg/mL 10 µg/disk 10 µg/disk

B. cereus 6.24 ± 0.08 9.67 ± 0.001 18.00 ± 0.086 20.67 ± 0.002 19.00 ± 0.002 NA
B. subtilis —­ —­ —­ —­ 18.00 ± 0.014 NA
S. aureus —­ —­ —­ —­ 20.00 ± 0.026 NA
S. typhimurium —­ —­ 7.86 ± 1.15 11.00 ± 1.00 26.00 ± 0.001 NA
S. mutans 11.50 ± 0.002 13.00 ± 0.042 14.25 ± 0.016 17.00 ± 0.012 24.00 ± 0.024 NA
C. albicans 7.24 ± 1.15 10.00 ± 1.00 12.33 ± 1.53 12.67 ± 1.53 NA 10.00 ± 0.046
S. cerevisiae 7.33 ± 0.58 9.33 ± 0.57 13.00 ± 0.01 17.68 ± 1.53 NA 02.50 ± 0.004
A.fumigatus —­ —­ —­ —­ NA 07.50 ± 0.012
A.niger —­ —­ —­ —­ NA 05.00 ± 0.054

Abbreviations: NA, not applicable; SD, standard deviation.

TA B L E 7 Minimum inhibitory concentration assay for Callistemon citrinus aerial part essential oil

Microbial strains MIC values (mg/mL) Essential oil MIC values (mg/mL) Ciprofloxacin MIC values (mg/mL) Fluconazole

B.cereus 2.00 02.50 NA

B. subtilis —­ 10.00 NA
S. aureus —­ 05.20 NA
S. typhimurium 4.50 07.50 NA
S. mutans 2.50 06.20 NA
C. albicans 4.00 —­ 05.00
S. cerevisiae 4.50 —­ 07.50
A. fumigatus —­ —­ 12.50
A. niger —­ —­ 12.50

Abbreviations: CCEO, Callistemon citrinus aerial part essential oil; NA, not applicable.

TA B L E 8 Anti-­cholinesterase activity of Callistemon citrinus aerial part essential oil and standard galanthamine

Concentrations for standard and Galanthamine % of CCEO % of IC50 value for IC50 value for
essential oil sample (µg/mL) inhibition ± SD inhibition ± SD Galanthamine CCEO

1 05.24% ± 0.0042 07.92% ± 0.0064 6.652 µg/mL 6.335 µg/mL

2 09.47% ± 0.0001 16.05% ± 0.0104
3 17.08% ± 0.0121 23.89% ± 0.0004
4 26.35% ± 0.0327 30.54% ± 0.0241
5 35.06% ± 0.0428 40.44% ± 0.0132
6 47.12% ± 0.0326 47.12% ± 0.0434

Abbreviations: %, percentage; CCEO, Callistemon citrinus aerial part essential oil; IC50, 50% inhibition; SD, standard deviation.

TA B L E 9 Allium cepa assay root growth test for Callistemon citrinus aerial part essential oil

Concentration (μg/mL) Root length before treatment (cm) Root length after treatment (cm) Total root growth length (cm)

Distilled water 6.25 cm ± 0.00213 7.64 cm ± 0.00242 01.39

Positive control EMS 6.16 cm ± 0.00412 6.42 cm ± 0.00120 00.26
CCEO 6.22 cm ± 0.01640 6.92 cm ± 0.00462 00.70

Abbreviations: CCEO, Callistemon citrinus aerial part essential oil; EMS, ethyl methanesulfonate; SD, standard deviation.
GOGOI et al. | 9 of 12

TA B L E 1 0 Mitotic index for Callistemon citrinus aerial part essential oil (Allium cepa assay)

Sample MI (%) P (%) M (%) A (%) T (%)

Distilled water 18.22 44.28 32.84 17.46 05.42

EMS 4.96 89.24 10.76 0 00.00
CCEO 17.25 51.60 28.99 13.15 06.26

Abbreviations: %, percentage; A, anaphase; CCEO, Callistemon citrinus aerial part essential oil; M, metaphase; MI, mitotic index; P, prophase; T,
telophase.

TA B L E 1 1 Chromosomal aberration test for Callistemon citrinus aerial part essential oil

Total
Compound Concentration Time Bridges Stickiness Clumped Multipolarity Breakage abberations

Distilled water 01.00 µg/mL 72 hr 4 3 0 1 2 2.00%

CCEO 6 5 2 4 3 4.00%
EMS 32 25 10 11 14 18.40%

Abbreviations: %, percentage; CCEO, Callistemon citrinus aerial part essential oil.

F I G U R E 2 Chromosomal aberrations used for the present investigation (genotoxicity test) for Callistemon citrinus aerial part essential
oil (Allium cepa assay): a. Chromosome Bridge, b. Chromosome Breakage, c. Multipolar Cell, d. Sticky Chromosome and e. Clumped
Chromosome

4 | D I S CU S S I O N compound of present investigation. C. citrinus essential oil possesses

wide applications as a cost effective, easy source for the isolation
Traditional applications of aromatic and medicinal plants are being of pure compound. There were few previous reports available that
investigated widely for their possible applications in the field of food, C. citrinus essential oil composes eucalyptol as the major compound
pharmaceutical, and aromatic industries. Eucalyptol is the major (Jamzad et al., 2014; Jazet et al., 2009; Opeoluwa et al., 2009;
10 of 12 | GOGOI et al.
Srivastava et al., 2001). Reports from Pakistan, Iran, Nepal, and
South Africa also showed eucalyptol as the major compound of C. cit-
rinus leaf essential oil (Oyedeji et al., 2009; Riaz & Chaudhary, 2011;
Shrestha et al., 2015; ZandiSohani et al., 2012). C. citrinus flowers es-
sential oils from Western Himalayas were also reported to be domi-
nated by 1, 8-­cineole (36.6%), followed by α-­pinene (29.7%) (Kumar
et al., 2015). Present investigation supports the aforesaid findings.
Although, the yield percentage of the major compounds varies in
all the previous studies, for example, South African CCEO showed
61.20% of 1,8-­cineole but present investigation showed 55.40%.
This difference of yield can be attributed to difference in generic
and geographical as well as environmental conditions (Oyedeji
et al., 2009). Consumption of antioxidants in human diets is essential
for controlling metabolic disorders such as diabetes as well oxidative-­
related inflammatory disorders. C. citrinus essential oil showed strong
antioxidant property and this activity may be due to the presence
of major compound eucalyptol and other minor constituents with
proven antioxidant potentials (Lee & Shibamoto, 2001). Current stud-
ies clearly depicted the antioxidant properties of CCEO and have the
prospective for future uses in food industries. Present findings are
supported by a previous report, where it was shown that due to the
presence of 1,8-­cineole the essential oil of C. citrinus possess antioxi-
dant activity with an IC50 value of 7.48 µg/mL (Mande & Sekar, 2020).
A report by Jamzad et al. (2014) showed that CCEO (8.58%) and hy-
dromethanolic extract of flowers (93.32%) possess antioxidant activ-
ity but best antioxidant activity was shown by flower extracts due to
higher phenolic compounds. The closely related species Melaleuca
citrina leaf essential oil was observed with IC50 value of 4.02 mg/mL
(Shukla et al., 2012); while Abdelhady and Aly (2012) reported radical
scavenging percentage 91.1 ± 0.3% for the same.
Strong anti-­inflammatory activity was also shown by the CCEO
and it may be due to the presence of major compound eucalyptol
(55.40%). There were evidences that eucalyptol and M. alternifolia
possess strong anti-­inflammatory activities (Chezet et al., 2006).
Except the aforesaid report, C. citrinus essential oil anti-­inflammatory
activity is not much known. However, there was a report by Radulovic
et al. (2015), where it was shown that a major constituent, a phlo-
roglucinol, 1-­(2,6-­dihydroxy-­4-­methoxyphenyl)-­3-­methylbutan-­1-­o
ne exhibits potent anti-­inflammatory activity in a dose-­dependent
manner. Present results make the essential oil worthy for further
anti-­inflammatory drug formulation and development.
In the present study, it was found that the antimicrobial activ-
ity against tested nine microbial strains varied at different concen-
trations. This is the first antimicrobial activity evaluation of CCEO
from North-­East India and as the MIC values were too high, the es-
sential oil could not be a promising source for natural antimicrobial
agent. Earlier, a study (Shukla et al., 2012) showed inhibition zones of
7.5 mm (E. coli), 9.2 mm (S. aureus), and 8.6 mm (B. cereus) by C. citrinus
essential oil. There was another study by Jazet et al. (2009) showed
that the essential oil fraction of C. citrinus rich in the eucalyptol and
linalool showed highest inhibition of growth toward Phaeoramularia
angolensis fungi. But a report from the Nepal showed that C. citrinus
leaf essential oil was not antimicrobial (MIC 625 µg/mL against A.
niger; 1,250 µg/mL against P. aeruginosa, and 2,500 µg/mL against E.
coli), larvicidal, or nematicidal (Shrestha et al., 2015). Reports from
South Africa (Oyedeji et al., 2009) and Brazil (Silva et al., 2010), also
showed that C. citrinus essential oils has marginally antibacterial ef-
fects. Present investigation says that C. citrinus essential oil does not
possess strong antimicrobial activity; it could be used as antimicro-
bial agent only in higher concentrations such as 2.5 mg/mL against
B. cereus, 4.5 mg/mL against S. typhimurium, 2.5 mg/mL against S.
mutans, and 4.0 mg/mL against C. albicans.
The CCEO also possess neurodegenerative inhibitory potential
and this was due to the presence of major compound eucalyptol. The
eucalyptol and limonene were previously reported for their excellent
AChE inhibitory activities (Chaiyana & Okonogi, 2012). Still, further-
more deep analysis is required to fully validate the potential of the
studied essential oil. Additionally, the CCEO did not show genotox-
icity in 1 µg/mL concentration. Furthermore, it was also confirmed
that eucalyptol does not possess any negative effects like carcinoge-
nicity, developmental toxicity, and mutagenicity on the experiments
performed on animals. Sub-­acute hepatotoxic and nephrotoxic ef-
fects in animal experiments appeared only at high oral dose in rats
weighing 2.5 g/kg (Kirsch & Buettener, 2013). The cytotoxicity of
C. citrinus essential oil was previously evaluated by sulforhodamine
B and MTT assay showed no toxicity in normal cells and reported
to be potential source of natural anti-­cancer compound for human
health (Kumar et al., 2015). Another cytotoxicity study by Shrestha
et al. (2015) showed that the essential oil of C. citrinus is ineffective
against MCF-­7 cells with only 17.50% kill at 100 µg/mL of essential oil
concentration and nematodes with LC50 of greater than 2,500 µg/mL.
Although, there were few reports regarding the pharmacokinetics,
safety of the compound eucalyptol (Bhowal & Gopal, 2015); but so far
there is no any previous report regarding the genotoxicity analysis of
CCEO, making this study scientifically more important.
5 | CO N C LU S I O N
Callistemon citrinus aerial part essential oil is rich in eucalyptol which
could be a cheap and easy source for the extraction of the pure com-
pound. The present study demonstrated the anti-­inflammatory, an-
tioxidant, antimicrobial, and anti-­cholinesterase activities indicating
that the essential oil can be considered a prospective source of new
drugs for the treatment of various inflammatory, oxidative, micro-
bial, and central nervous system (CNS) disorders as well as for the
application in cosmetic, food, and beverages industries in near fu-
ture. From a toxicological point of view CCEO was found to be safe
due to negligible genotoxicity. However, further studies are going on
to obtain purified compounds that may be responsible for the activi-
ties observed from the C. citrinus essential oil sample.
AC K N OW L E D G M E N T S
The authors are thankful to the Director of CSIR-­NEIST, Jorhat
Assam for providing us laboratory facilities for conducting the re-
search activity.
GOGOI et al.
C O N FL I C T O F I N T E R E S T
The authors declare that they have no conflict of interest.
AU T H O R C O N T R I B U T I O N S
Roktim Gogoi: Conceptualization; Data curation; Investigation;
Software; Writing-­original draft. Twahira Begum: Formal analysis;
Validation; Writing-­review & editing. Neelav Sarma: Formal analy-
sis; Methodology; Validation. Sudin Kumar Pandey: Formal analysis;
Investigation; Validation. Mohan Lal: Conceptualization; Project ad-
ministration; Resources; Writing-­review & editing.
DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were gener-
ated or analyzed during the current study.
ORCID
Mohan Lal https://orcid.org/0000-0001-6995-3377
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