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R E S E A R C H A R T I C L E Open Access
Standardized ethanol extract, essential oil
and zerumbone of Zingiber zerumbet
rhizome suppress phagocytic activity of
human neutrophils
Nabilah Mohammad Yaqoob Akhtar
1
, Ibrahim Jantan
2*
, Laiba Arshad
3
and Md. Areeful Haque
4
Abstract
Background: Zingiber zerumbet rhizome and its bioactive metabolites have previously been reported to exhibit
innumerable pharmacological properties particularly anti-inflammatory activities. In the present study, the 80%
ethanol extract, essential oil and zerumbone of Z. zerumbet rhizomes were explored for their in vitro
immunosuppressive properties on chemotaxis, CD11b/CD18 expression, phagocytosis and chemiluminescence of
isolated human polymorphonuclear neutrophils (PMNs).
Methods: The extract was analyzed quantitatively by performing a validated reversed phase high performance
liquid chromatography (RP-HPLC). Zerumbone was isolated by chromatographic technique while the essential oil
was acquired through hydro-distillation of the rhizomes and further analyzed by gas chromatography (GC) and GC-
MS. Chemotaxis assay was assessed by using a 24-well cell migration assay kit, while CD18 integrin expression and
phagocytic engulfment were measured using flow cytometry. The reactive oxygen species (ROS) production was
evaluated by applying lucigenin- and luminol-enhanced chemiluminescence assays.
Results: Zerumbone was found to be the most abundant compound in the extract (242.73 mg/g) and the oil
(58.44%). Among the samples tested, the oil revealed the highest inhibition on cell migration with an IC
50
value of
3.24 μg/mL. The extract, oil and zerumbone showed moderate inhibition of CD18 integrin expression in a dose-
dependent trend. Z. zerumbet extract showed the highest inhibitory effect on phagocytic engulfment with
percentage of phagocytizing cells of 55.43% for PMN. Zerumbone exhibited strong inhibitory activity on oxidative
burst of zymosan- and PMA-stimulated neutrophils. Zerumbone remarkably inhibited extracellular ROS production
in PMNs with an IC
50
value of 17.36 μM which was comparable to that of aspirin.
Conclusion: The strong inhibition on the phagocytosis of neutrophils by Z. zerumbet extract and its essential oil
might be due the presence of its chemical components particularly zerumbone which was capable of impeding
phagocytosis at different stages.
Keywords: Zingiber zerumbet, Zerumbone, Essential oil, Immunosuppressive effects, Phagocytic activity, Human
neutrophils
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: profibj@gmail.com
2
School of Pharmacy, Faculty of Health and Medical Sciences, Taylor’s
University, Lakeside Campus, 47500 Subang Jaya, Selangor, Malaysia
Full list of author information is available at the end of the article
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331
https://doi.org/10.1186/s12906-019-2748-5
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
The immune system is a sophisticated network of subsys-
tems involving the coordination of various cells, proteins
and chemical signals against infectious diseases. This pre-
eminent system is classified into innate immunity (non-
specific) and adaptive immunity (acquired or specific).
Phagocytosis is the host’s defense mechanism which acts
as the essential component of a carefully orchestrated cas-
cade of events in the innate immunity. Professional phago-
cytes like neutrophils, macrophages and monocytes are
the main line of defense which perform various functions
in an inflammation or immune responses. These functions
include interacting, identifying, capturing foreign particles
and eliminating pathogens which invades the body. The
most plentiful type of leukocytes residing in the blood are
the polymorphonuclear neutrophils (PMNs) which are the
earliest to migrate from the blood to infected sites for
eradicating pathogens and removing cellular debris [1].
Predominantly, there are four steps in the phagocytosis
process involving the phagocytes, namely chemotaxis, ad-
hesion, engulfment and degradation via respiratory burst.
Upon the invasion of pathogenic micro-organisms, foreign
particles and events in the human body, the initial re-
sponse in the first few hours plays a significant and critical
role which is responsible for the consequence of the infec-
tion [2].
In a healthy individual, the activation of the immune sys-
tem as a defense mechanism demonstrates the capability of
maintaining homeostasis in the body. However, uncon-
trolled reactions resulting from impaired immune system
functions can lead to tissue damage and disorders including
hypersensitivity (overactive immune response), immunodefi-
ciency (ineffective immune response) and autoimmunity
(improper reaction to self) [2,3]. Many immunostimulants
and immunosuppressants in current clinical uses have major
limitations due to their cytotoxicity causing severe adverse
effects including nephrotoxicity, hepatotoxicity, hyperten-
sion, gastrointestinal toxicity, metabolic toxicity, and affect-
ing rapidly growing cells [4]. Due to this setback, the usage
of plant-derived herbal medicines and compounds is gaining
interest among researchers in the development of safer and
potent immunomodulating agents [5,6]. Compounds with
immunomodulating potential usually come from plants’sec-
ondary metabolites including flavonoids, isoflavonoids, phy-
tosterols, sesquiterpenes, indoles, polysaccharides, alkaloids,
tannins and glucans [7,8].
Zingiber zerumbet (L.) Roscoe ex Sm. (Family: Zingi-
beraceae) is widely distributed in all tropical regions es-
pecially in Southeast Asia, Pacific and Oceania. The
rhizomes of the plant have been consumed as spices and
used traditionally to treat various immune-inflammatory
related disorders [9]. Numerous compounds have been
isolated from Z. zerumbet which serve as potent and de-
pendable medicinal candidates for innumerable disorders.
Among the compounds, the most isolated and utilized bio-
active metabolite is zerumbone [10–12]. Previous studies
indicated that the plant possessed many pharmacological
activities including immunomodulatory, anti-inflammatory,
antioxidant, antinociceptive, anticancer and antibacterial
[13–15]. Recently, we reported that Z. zerumbet extract
(ZZE) and zerumbone (ZER) demonstrated inhibitory ef-
fects against inflammation and related disorders pertaining
totheimmunesystemthroughthesuppressionofseveral
pro-inflammatory markers via the MyD88-dependent NF-
κB, MAPKs, and PI3K-Akt activation [9,11]. The present
study was the first to be performed in determining the ac-
tivity of the standardized extract of Z. zerumbet including
its essential oil (ZZEO) and marker compound, ZER on the
four steps of phagocytosis in human neutrophils.
Methods
Chemicals and reagents
Serum opsonized zymosan A (Saccharomyces cerevisiae sus-
pensions and serum), lipopolysaccharide (LPS), lucigenin
(10,10′-dimethyl-9,9′-biacridinium, dinitrate), luminol (3-
aminophthalhydrazide), Hanks Balance Salt Solutions
(HBSS), fluorescein isothiocyanate (FITC)-labelled opson-
ized Escherichia coli, trypan blue, ibuprofen (purity 99%),
acetylsalicylic acid (purity 99%), phorbol 12-myristate 13-
acetate (PMA), phosphate buffer saline tablet (PBS), and
dimethylsulfoxide (DMSO) were acquired from Sigma (St
Louis, MO, USA). ZER (Sigma, St Louis, USA) standard
with 98% purity was used as marker compound for quantita-
tive determination of compounds present in the extract by
high performance liquid chromatography (HPLC). Chemilu-
minescence measurements were performed on a Luminos-
kan Ascent luminometer (Thermo Scientific, UK). RPMI-
1640, fetal bovine serum (FBS), cytoselect 24-well cell migra-
tion assay kits, penicillin, streptomycin were purchased from
Cell Biolabs, Inc. (CA, USA). The phagotest kit was pro-
cured from Glycotope Technology, Germany. Immuno-
globulin G-FITC, FITC-conjugated CD18, APC-conjugated
CD11b and FACS lysing solution were acquired from BD
Biosciences, USA. HPLC grade methanol and acetonitrile
were purchased from E-Merck. Dichloromethane was used
as a solvent. The essential oil obtained from hydro-
distillation was dried with anhydrous MgSO
4
. A HPLC (Wa-
ters 2998) (Leitz Watzler, Germany), light microscope, a
CO
2
incubator (Shell Lab, USA), and a flow cytometer
BDFACS Canto II equipped with 488 nm argon-ion laser
were also utilized.
Preparation of extract and isolation of zerumbone
The whole plant of Zingiber zerumbet was obtained from
Kuantan, Pahang, Malaysia in November 2016. The plant
material was identified by a botanist, Dr. Abdul Latif
Mohamad, at the Faculty of Science and Technology,
Universiti Kebangsaan Malaysia (UKM), Malaysia and a
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 2 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
voucher specimen (no: UKMHF137) was deposited at
the Herbarium of UKM. The rhizomes of Z. zerumbet
(1.75 kg) were ground, dried and macerated using 3 L of
80% ethanol (3 times) for 72 h at room temperature be-
fore being filtered by Whatman No.1 filter paper (What-
man, England). The filtrates were then pooled, collected
and any residual solvent was removed by a rotary evap-
orator at 40 °C to obtain a dark brown extract. The ex-
tract was subsequently freeze-dried to acquire a crude
gummy-like extract with a yield of 14.7% and stored at
4 °C for further use [9,11]. ZER was isolated according
to the method of Haque et al. [11]. Briefly, the concen-
trated crude extract (10 g) was subjected to repeated col-
umn chromatography (40–63 μm, 3 × 60 cm) with n-
hexane: ethyl acetate (10,0–7:3 ratios, v/v). The eluates
collected were allowed to evaporate slowly from the solv-
ent for re-crystallization. Upon repeated recrystallization
from n-hexane-ethyl acetate, white crystals of 87.4 mg of
ZER (0.87%) were obtained. The purity (> 98%) and iden-
tity of ZER were confirmed based on ESI-MS and NMR
spectroscopy and its physicochemical property [11].
Figure 1shows the chemical structure of ZER and Fig. 2
depicts
13
C-NMR spectrum of ZER. Additional file 1:Fig.
S1 and Additional file 2:Fig.S2show
1
HNMR and
HRESI-MS spectra of the compound.
Preparation of essential oil
The fresh Z. zerumbet rhizomes were cleaned, cut into
small pieces and dried under shade for three days. The
dried material (2.5 kg) was subjected to hydrodistillation
for 8 h in a Clevenger-type apparatus (WUTEG, Germany)
to obtain 9.2 mL of light yellowish oil. The oil was dried
over anhydrous magnesium sulphate to remove traces of
moisture and kept at 4 °C until further use.
GC and GC-MS analysis of the essential oil
The analysis of Z. zerumbet essential oil (ZZEO) was
performed by the Shimadzu GC-2010 with column DB-5
(30 m × 0.25 mm i.d, 1.0 μm film thickness) equipped
with a flame ionization detector (FID). The oil was dis-
solved in ethyl acetate and automatically injected in split
mode with nitrogen as the carrier gas at a pressure of
50.0 mL/min at a flow rate of 1.19 mL/min. The initial
column temperature of the oven, set at 75.0 °C for 10
min was gradually increased to 250 °C at the rate of
3 °C/min for 5 min. A homologous series of n-alkane
standards (C9 to C22) were additionally subjected within
the same condition as the essential oil. The linear reten-
tion indices (RI) were calculated corresponding to the n-
alkane standards [16,17]. The essential oil was also ana-
lyzed by GC-MS performed on an Agilent 7890A gas
chromatograph (GC) directly coupled to the mass spec-
trometer system (MS) of an Agilent 5975C inert MSD
with triple-axis detector. The model used was DB-5MS-
UI (30 m × 0.25 i.d, 0.25 μm thickness) with helium as
the carrier gas at a flow rate of 1.3 mL/min. The
temperature of the oven, initially programmed at 75.0°C
for 10 min was increased gradually at 3 °C/min to 250 °C
and held for 5 min. Peak identification in the GC chro-
matogram was carried out based on the MSD Chemsta-
tion and a library search was performed for all peaks by
the NIST/EPA/NIH version 2.0 database (Agilent tech-
nologies). The compounds were also identified by com-
parison of calculated retention indices with literature
values and co-chromatography of some constituents with
authentic components on the DB 5 capillary column.
HPLC analysis for standardization of 80% ethanol extract
of Zingiber zerumbet rhizome
Standardization of the plant extract has been performed
as stated by Haque et al. [9]. Briefly, 3 mg of Z. zerumbet
80% ethanol extract and 1 mg of the reference standard
(ZER) were dissolved in 1 mL of methanol and sonicated
for 10 min. The stock solutions were filtered through
0.45 μm nylon filter membrane (Maidstone, Kent, UK).
ZER (98% purity) was purchased from Sigma, St. Louis,
USA. Thereafter, the diluted solutions of the reference
standards and extracts were analyzed using HPLC under
the subsequent settings: column: reversed phase, C-18
column (250 mm × 4.6 mm i.d, 5 μm, Xbridge, Waters,
Ireland), and detector: PDA (Waters 2998) with injection
volume 20 μL and wavelength 250 nm. Gradient elution
method was employed using solvent A (acetonitrile) and
solvent B (water) as the mobile phase at a flow rate of
1.2 mL/min for the analysis of the standard compound
and extract. The initial composition for mobile phase
was 65% solvent A, then increasing to 70% of solvent A
over 10 min followed by 75% solvent A and held for 16
min. Compound identification in the extract sample was
Fig. 1 Chemical structure of zerumbone
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 3 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
done via comparison of the retention times and peak
spectra of those acquired from the standard. Quantifica-
tion of compound in the extract was determined from
the standard curve equation plotted from four concen-
trations of the standard solution.
Validation of HPLC method
HPLC method was validated by determination of the
precision, linearity, limits of detection (LOD) and quan-
tification (LOQ). Linearity was determined from the cor-
relation coefficient (r
2
) obtained based on the calibration
curve plotted from a range of concentrations of 125 to
1000 μg/mL of the standard. The precision of the HPLC
method on repeatability and intermediate precision were
computed as the relative standard deviation (RSD) from
the injection of standard samples ranging from concen-
trations of 125 to 1000 μg/mL. Each concentration was
injected thrice per day (intraday precision) and on three
separate days (interday precision). LOD and LOQ were
calculated from the RSD and slope (S) of the calibration
curve by using the following equation: LOD =
3.3 × (RSD/S) and LOQ = 10 × (RSD/S).
Isolation of human polymorphonuclear neutrophils
Fresh blood was attained by aseptic vein puncture as de-
scribed by Arshad et al. [18] from healthy volunteers
who were non-smokers, fasted overnight and not con-
suming any supplements or medications. Briefly, 10 mL
whole blood with equal amount of HBSS were allowed
to sediment for 30 min at room temperature. The sepa-
rated plasma layer was layered onto lymphoprep gradi-
ent (1077 mg/mL) and centrifuged at 400 × g for 20 min
at room temperature to allow neutrophils and erythro-
cytes settle at the bottom of the lymphoprep layer. One
millilitre of cold distilled water was briefly added for red
blood cells lysis followed by PBS before the mixture was
centrifuged at 300 × g at 4 °C for 10 min. Next, the
supernatant was carefully aspirated and the sedimented
pellet was suspended with HBSS for cell purification to a
final concentration of 1 × 10
6
cells/mL. The Human
Ethical Committee of Universiti Kebangsaan Malaysia
(Approval no: UKM PPI/111/8/JEP-2017-335) approved
the use of human blood in this study. Volunteers who
participated in this study provided written informed
consent for blood collection.
Cell viability
The cells were subjected to viability test using trypan
blue exclusion method as described by Jantan et al. [19]
to determine the cytotoxicity of samples. Briefly, 200 μL
of ZZE and ZZEO (3.13 to 100 μg/mL) and ZER at 0.63
to 20 μg/mL (3.13 to 50 μM) were incubated in 5% CO
2
Fig. 2
13
C NMR spectrum of zerumbone
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 4 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
incubator at 37 °C for 2 h with equal volume of cell sus-
pensions (1 × 10
6
cells/mL) in triplicates. After incuba-
tion, 20 μL of the mixture was mixed with 20 μL trypan
blue. The blue dye uptake indicated cell death and cell
viability percentage was determined with the aid of
hemocytometer.
Chemotaxis assay
The inhibitory effect of the test samples towards PMN
chemotaxis was measured as described by Arshad et al.
[20] with slight adjustments. Chemotaxis assay was con-
ducted by using Cytoselect 24-well cell migration kit
based on the protocol set by the manufacturer (Cell Bio-
labs Inc.). The assay started off by adding 500 μLof
RPMI media comprising 10% FBS as chemoattractant
into the lower chamber. The cell suspension was pre-
pared in serum-free media to a final concentration of 1.5
×10
6
cells/mL. The upper chamber containing polycar-
bonate membrane inserts with 3 μm pore size filters was
filled with 300 μL of cell suspension mixed with test
samples ranging from five serial dilution concentrations
(ZZEO and ZZE: 40 to 2.5 μg/mL and ZER: 10 to
0.63 μg/mL). The control wells consist of RPMI with
10% FBS and cells without any test samples. Ibuprofen
was used as the positive control in relation to an earlier
study demonstrating ibuprofen, a very efficacious NSAID
in obstructing the migration of PMNs [21]. The 24-well
tissue culture plate was allowed to incubate for 2.5 h in a
CO
2
incubator at 37 °C to allow cell migration towards
chemoattractant. The migrated cells sifted across the
polycarbonate membrane and adhere at the surface
underneath. The inserts were subsequently shifted to a
clean well of 200 μL cell detachment buffer to be incu-
bated for 30 min at 37 °C in CO
2
incubator to allow cells
to detach from the bottom of the membrane inserts.
After incubation, the inserts were gently tilted inside the
cell detachment solution to dislodge cells before the in-
serts were finally discarded. The migratory cells were
lysed and stained by the addition of lysis buffer and
CyQuant® GR Fluorescent Dye. Finally, the migrated
cells were quantified by determination of fluorescence as
detected by Victor 2 plate reader (Perkin Elmer, Inc.)
CD11b/CD18 integrin (mac-1) expression assay
The method was carried out as explained by Harun et al.
[22] with slight amendments. Aliquots of 100 μL of hep-
arinized whole blood and 20 μL of each test sample were
incubated at three respective concentrations (ZZEO and
ZZE: 50 to 3.13 μg/mL and ZER: 10 to 0.63 μg/mL) in
5% CO
2
for 30 min at 37 °C. Control tubes did not con-
tain any test samples. The sample mixture was stimu-
lated with LPS (0.25 μg/mL) and again incubated for 1.5
h. The reaction was then brought to a halt by concur-
rently transferring the tubes onto ice. Ten μL of APC-
conjugated CD11b and FITC-conjugated CD18 as well
as 10 μL of IgG-FITC (negative control) was further
added and all tubes were incubated for 1 h on ice.
Thereafter, FACS lysing solution was added and the
mixture was incubated in the dark for red blood cells
lysis for 20 min before the tubes were centrifuged at 250
× g at 4 °C for 5 min. The supernatant was aspirated and
cells were recurrently washed twice with PBS. Finally,
cells were suspended in 500 μL PBS before being ana-
lysed by flow cytometer to evaluate the expression of ad-
hesion molecules. The mean fluorescence intensity of
antibody-stained cells was recorded as percentage ex-
pression of CD11b and CD18.
Phagocytosis assay
Phagocytic activity was evaluated by performing the
assay based on the manufacturer’s protocol with the
Phagotest assay kit (Glycotope Technology, Germany).
In brief, 100 μL heparinised peripheral whole blood with
20 μL of test samples at three respective concentrations
(ZZEO and ZZE: 50 to 3.13 μg/mL; ZER: 10 to 0.63 μg/
mL) and 20 μL FITC-labelled E.coli at 37 °C was incu-
bated in a closed shaking water bath at 60 rpm for 30
min, with the negative control remaining on ice. Cells
without samples and engulfment activity at 37 °C was
used as positive control. Following incubation, all tubes
were simultaneously shifted onto an ice box followed by
addition of 100 μL of ice-cold quenching solution to
quench phagocytosis. After 3 mL of washing solution
was added, the tubes were centrifuged at 250 × g (4 °C)
for 5 min and the supernatant was discarded. Two mL of
lysing solution was added after washing twice and
followed by incubation in the dark for 20 min (37 °C).
After incubation, the tubes were centrifuged at the same
speed and cells were lastly resuspended in 200 μLof
DNA staining solution. The phagocytic activity was ana-
lyzed via flow cytometry as the percentage of E. coli en-
gulfment by phagocytizing neutrophils.
Chemiluminescence assay
Chemiluminescence assay was assessed as explained by
Jantan et al. [19]. Briefly, 25 μL of diluted whole blood in
PBS (1:50) or 25 μL PMN suspended in HBSS was incu-
bated (at 37 °C for 30 min) with 25 μL test samples at
different serial dilution concentrations (ZZEO and ZZE:
40 to 2.5 μg/mL; ZER: 10 to 0.63 μg/mL) in 96-well flat
bottom microplates. The DMSO content in the mixture
was altered to a final concentration of 0.6% to exclude
solvent effect for chemiluminescence. Luminol, cells,
0.6% DMSO and HBSS++ acted as negative control
while 25 μL aspirin as positive control. Cells were then
stimulated by 25 μL serum opsonised zymosan (SOZ)
followed by 25 μL of luminol as a probe, or 25 μL phor-
bol 12-myristate 13-acetate (PMA) followed by 25 μL
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 5 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
lucigenin. The final volume in each well was adjusted
with HBSS to 200 μL. Thereafter, the microplates were
incubated in a thermostatically controlled chamber of a
luminoskan at 37 °C for 50 min. The readings shown
were identified as reading luminoskan unit (RLU). The
percentage of inhibition was calculated from the formula
as follows:
Inhibition %ðÞ¼RLUcontrol RLUsample
x 100%
RLUcontrol
Statistical analysis
The results were represented as means ± standard error
of the mean (SEM) of the data obtained from triplicate
experiments. The IC
50
values of test samples were evalu-
ated by Graph Pad Prism 5 Software based on at least
three determinations. Statistical analysis was performed
via one-way analysis of variance (ANOVA) for multiple
comparisons followed by Dunnet’s post hoc test using
Statistical Analysis software SPSS11.5, and p< 0.05 was
regarded as statistically significant.
Results
Analysis of the components of essential oil
Hydrodistillation of Z. zerumbet rhizomes yielded 0.37%
of essential oil. The GC analysis of the essential oil iden-
tified 17 compounds. Based on Table 1, the main com-
pound identified was 2,6,10-cycloundecatrien-1-one, 2,6,
9,9-tetramethyl-, also known as zerumbone (ZER) which
constituted 58.44% of the oil. The relative amounts of
individual components were based on peak areas ob-
tained, without FID response factor correction.
Quantification of chemical marker using RP-HPLC
According to our earlier report, the chromatograms ob-
tained from the reversed-phase HPLC column of the
80% ethanol extract of Z. zerumbet revealed several
peaks with zerumbone as the major peak, at retention
time of 9.745 min (Additional file 3: Fig. S3) [9]. Peak
identification was performed by comparison with HPLC
of the reference standard, ZER. The plotted calibration
curves showed linearity corresponding to the correlation
coefficient (r
2
) of 0.999 over a range of concentration
from 125 to 1000 μg/mL. The reproducibility of the
HPLC result was demonstrated by good precision from
the method employed conforming to the %RSD values
obtained as illustrated by the small values of standard
deviation for retention time and responses of the marker
compounds for both intraday and interday assay preci-
sions. The %RSD for interday and intraday assay preci-
sions was analyzed as 0.93 and 1.53% disparately in
respect to the retention time while 0.57 and 5.92% cor-
respondingly in the case of peak area. The limit of detec-
tion (LOD) and limit of quantification (LOQ) for ZER
was 0.117 and 0.355 μg/mL, respectively. The small LOD
and LOQ values established that the method used exhib-
ited good sensitivity. The quantitative determination of
Table 1 Percentage composition of essential oil of Zingiber zerumbet
Compound Percentage composition (%) Kovat Index
c
Method of Identification
α-Pinene 1.27 939 a,b,c
Camphene 5.36 957 a,b,c
α-Terpinene 0.36 1018 a,b,c
1,8-Cineole 1.70 1032 a,b,c
β-Ocimene 1.46 1038 a,b,
Nonen-1-ol 1.75 1158 a,b,
Terpinen-4-ol 1.53 1178 a,b,c
β-Caryophyllene 0.92 1420 a,b,c
α-Humulene 12.24 1458 a,b,c
(E)-Nerolidol 1.44 1564 a,b
Caryophyllene oxide 2.05 1581 a,b
Humulene epoxide 4.96 1608 a,b
Caryophylla-4 (14),8 (15)-dien-5.alpha.-ol 3.86 1642 a,b
Calamenene 0.49 1662 a,b
α-Bisabolol 1.03 1674 a,b
Farnesol 0.81 1708 a,b
Zerumbone 58.44 1734 a,b,c
a: analysis by mass fragmentation pattern in gas chromatography mass spectrometry (GC-MS)
b: Kovat indices on a DB5 column (1 μm thickness, 30.0 m length, 0.25 mm diameter)
c: co-chromatography with authentic sample
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 6 of 12
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the major compound revealed ZER as the main constitu-
ent found in Z. zerumbet extract, which was calculated
to be 242.73 mg/g.
Cell viability assay
The cytotoxicity of ZZE, ZZEO and ZER from Z. zerum-
bet was evaluated on human whole blood and PMNs.
After the cells were subjected to 2 h of incubation with
test samples, the concentrations at which the cells were
found viable ≥90% were below 50 μg/mL for ZZE and
ZZEO, and 10 μg/mL (50 μM) for ZER, suggesting the
samples were non-toxic for subsequent immunomodu-
lating assays at these concentrations.
Chemotaxis assay
The effect of ZZE, ZZEO and ZER on PMN migration is
shown in Fig. 3. As shown in the figure, all samples re-
vealed a dose-dependent inhibitory effect. ZZEO showed
the highest inhibitory activity on PMN chemotaxis with
IC
50
value of 3.24 μg/mL, comparable to the positive
control, ibuprofen with an IC
50
value of 1.70 μg/mL
(8.25 μM). The second highest inhibition was shown by
ZZE followed by ZER with IC
50
values of 4.83 and
6.21 μg/mL (28.44 μM), respectively.
CD11b/CD18 integrin (mac-1) expression assay
The inhibitory effects of ZZE, ZZEO and ZER were ana-
lyzed on the expression of Mac-1 by using flow cyt-
ometer. As shown in Table 2and Fig. 4,ZER at its
highest concentration (10 μg/mL) was the most potent
sample in suppressing CD11b/CD18 surface expression
with percentage expression 72.57% as compared to the
untreated sample (positive control). ZZE and ZZEO both
exhibited weak inhibition towards CD11b/CD18
expression on PMNs. All three samples of ZZE, ZZEO
and ZER demonstrated a dose-dependent trend of inhib-
ition in this assay.
Phagocytosis assay
The engulfment of opsonized E. coli by PMNs was eval-
uated using phagotest kit and analyzed by flow cytome-
try. The engulfment inhibitory activity at 37 °C was used
as a positive control and normal condition at 0 °C as a
negative control. Based on results as shown in Table 3
and Fig. 5,at50μg/mL the highest inhibition of phago-
cytic activity was shown by ZZE which exhibited the
highest engulfment inhibitory activity with percentage
phagocytizing cells of 55.43%, followed by ZZEO at
69.20%. ZER at 10 μg/mL showed percentage of phago-
cytizing cells of 76.97%.
Chemiluminescence assay
Preliminary screening was performed on the whole
blood to investigate the effects of samples on respiratory
burst upon activation by zymosan and PMA as illus-
trated in Table 4. The presence of intracellular ROS was
detected by luminol, whereas lucigenin was used to de-
tect extracellular ROS. For extracellular ROS production
in whole blood induced by PMA, ZER showed an IC
50
value of 6.83 μg/mL (31.25 μM) which was lower than
that of aspirin with an IC
50
value of 7.65 μg/mL
(42.46 μM). The samples were then further investigated
for their effects towards ROS production in PMNs. For
intracellular ROS production induced by zymosan, ZZE
showed an IC
50
value of 2.89 μg/mL, comparable to the
positive control, aspirin (1.95 μg/mL). ZER remarkably
inhibited extracellular ROS production in PMNs with an
IC
50
value of 3.79 μg/mL (17.36 μM), comparable to the
Fig. 3 Percentage inhibition of test samples on PMN chemotaxis presented as mean ± SEM (n= 3). Data were analyzed by one-way ANOVA
followed by Dunnet’s post hoc test. Significance of differences with respect to control: *p< 0.05
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 7 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
IC
50
value of aspirin which is 2.94 μg/mL (16.30 μM),
suggesting the immunosuppressive potential of ZER.
Discussion
Inhibition of migration of phagocytes to the site of infec-
tion upon activation by endogenous or exogenous che-
moattractants can account for part of the anti-
inflammatory activity of plant samples. The chemotaxis
assay indicated that ZZEO was the most potent in inhi-
biting PMN chemotaxis comparable to the positive con-
trol, ibuprofen. The anti-inflammatory activity of Z.
zerumbet is well supported by many previous studies.
The methanol extract of Z. zerumbet had significantly
inhibited the activities of cyclooxygenase (COX), lipoxy-
genase (LOX), myeloperoxidase (MPO) and nitric oxide
synthase (iNOS) (lipopolysaccharide-induced) [23]. Also
recent studies have shown that ZER significantly sup-
pressed p38 MAPK, an enzyme crucial for neutrophil
chemotaxis in LPS-stimulated macrophages [10,24],
thus may explain the inhibitory chemotactic migration
effect as observed in this assay. In addition, GC and GC-
MS analysis of the oil revealed the presence of the mono-
cyclic sesquiterpene, ZER as the major compound along
with other compounds. α-Pinene present in the oil has
been shown in previous study to significantly reduce the
migration of neutrophils reacting to chemoattractants,
Table 2 Percentage of CD11b/CD18 expression activity (%) of neutrophils at different concentrations of test samples derived from
Zingiber zerumbet
Sample (μg/mL) Concentration (μg/mL)
50 12.5 3.13
Z. zerumbet extract (ZZE) 87.67 ± 0.59 89.80 ± 0.66 96.23 ± 1.63
Z. zerumbet essential oil (ZZEO) 91.03 ± 0.63 92.93 ± 1.45 96.10 ± 0.85
10 2.5 0.63
Zerumbone (ZER) 72.57 ± 6.45* 76.47 ± 4.80* 86.77 ± 1.77
Positive control 92.47 ± 3.38
Data are presented as mean ± SEM, n= 3. Data were analyzed by one-way ANOVA followed by Dunnet’s post hoc test. Significance of differences with respect to
control: *p< 0.05
Fig. 4 CD11b/CD18 expression activity by neutrophils treated with test samples i.e., (a) positive control, (b)Z. zerumbet extract (50 μg/mL), (c)Z.
zerumbet essential oil (50 μg/mL), (d) zerumbone (10 μg/mL)
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 8 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
fMLP and LTB4 [25]. Consistently, ZZEO in our present
study revealed the presence of α-pinene which may have
contributed synergistically to produce a more pronounced
effect when compared to zerumbone as a single com-
pound. Various essential oils have demonstrated notable
anti-inflammatory reaction by obstructing leukocyte mi-
gration towards the inflammatory focus [26,27].
The binding of human leukocyte integrins (CD11a/
CD18, CD11b/CD18 and CD11c/CD18) to LPS triggers
deleterious systemic inflammatory responses when re-
leased into blood circulation, causing organ damage
[28]. The inhibition of CD11b/CD18 expression could
be due to the inhibition of adhesion molecule expres-
sions. Lipid A-like molecules were capable of deterring
the stimulatory effect induced by LPS, thus reducing the
upregulation of CD11b/CD18 surface expression and re-
duced the progress of inflammatory processes [28]. The
CD11b/CD18 integrin expression assay showed that ZER
was the most active in suppressing LPS binding site on
human leukocytes. ZER has been shown to suppress NF-
ĸB signaling pathway [10], which regulates various im-
mune and inflammatory genes expression involving cy-
tokines and adhesion molecules [29]. Thus this may
suggest that ZER was able to inactivate the expression of
CD11b/CD18 integrins via NF-ĸB pathway.
The key receptors in phagocytosis are the Fc receptor
and complement CR3 receptor. Fc-gamma receptors sense
immunoglobulin-contained particles while complement
Table 3 Percentage of phagocytic activity (%) of neutrophils at different concentrations of test samples derived from Zingiber
zerumbet
Sample (μg/mL) Concentration (μg/mL)
50 12.5 3.13
Z. zerumbet extract (ZZE) 55.43 ± 0.77* 58.50 ± 1.15* 59.30 ± 0.68*
Z. zerumbet essential oil (ZZEO) 69.20 ± 0.74* 70.83 ± 1.48* 77.87 ± 1.18*
10 2.5 0.63
Zerumbone (ZER) 76.97 ± 1.15* 77.73 ± 2.48* 79.70 ± 3.08*
Positive control 93.03 ± 1.56
Data are presented as mean ± SEM, n= 3. Data were analyzed by one-way ANOVA followed by Dunnet’s post hoc test. Significance of differences with respect to
control: *p< 0.05
Fig. 5 E. coli engulfment by neutrophils treated with test samples i.e., (a) negative control, (b) positive control, (c)Z. zerumbet extract (50 μg/mL),
(d)Z. zerumbet essential oil (50 μg/mL), (e) zerumbone (10 μg/mL)
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 9 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
receptors seek the particles opsonized by complement fac-
tors [30]. Fc receptors are expressed on neutrophils and
functions to phagocytose and intracellular killing of op-
sonized pathogens. Fc receptor’s responses can be
obstructed by ROS inhibitors and inhibitors of the H
2
O
2
-
myeloperoxidase-chloride system [31]. The results of this
study indicated that ZZE has the highest inhibition of
phagocytic activity. The results illustrate the inhibition of
complement opsonized E. coli uptake may result from the
suppression of the above-mentioned receptors on the
PMNs by the plant samples.
Upon stimulation by opsonized SOZ or PMA, PMNs
produced ROS which is generated through the NADPH
oxidase complex. The plant samples were investigated
for their inhibitory effects on the oxidative burst. Plant
constituents possessing antioxidant properties demon-
strate suppressive effect towards free radicals during oxi-
dative burst. In this study, ROS production was
determined upon stimuli with opsonized SOZ which
stimulates neutrophils via surface complement receptor
(CR3) and PMA which crosses the cellular membrane
and binds to protein kinase C independent of cell recep-
tor interaction [32]. The high inhibitory activity shown
by ZZE in luminol-amplified chemiluminescence is con-
sistent with the results of previous study which studied
the antioxidant activities of Z. zerumbet ethanol extract
on hydroxyl radical scavenging assays and DPPH and
demonstrating substantial radical scavenging activities
due to high polyphenol, flavonoid and kaempferol con-
tents in the extract [33]. ZER possesses an α,β-
unsaturated carbonyl group in its molecule and was ef-
fective in inhibiting PMA-induced oxidative burst. This
finding is in accordance with Arshad et al. [7] where the
α,β-unsaturated carbonyl moiety of ZER might act as an
antioxidant and was capable of being radical scavengers
via covalent ligand binding to target proteins.
Conclusion
The outcome of the current study corroborated ZER as
the major compound through HPLC quantitative and
qualitative analysis of ethanol extract of Z. zerumbet.
The hydrodistillation of essential oil of Z. zerumbet
similarly revealed zerumbone as its major constituent.
ZZEO showed the highest inhibitory effect followed by
ZZE in chemotaxis assay. Meanwhile, for phagocytic en-
gulfment and intracellular ROS production, ZZE showed
the highest inhibitory effect whereas, extracellular ROS
production was highly suppressed by ZER. Even though
ZZE and ZZEO contained other constituents, ZER was
the major contributor in inhibiting the respiratory burst
stage. Correspondingly, other secondary metabolites
present in ZZE and ZZEO might have acted synergistic-
ally with ZER, contributing towards the inhibitory ef-
fects. Therefore, the ethanol extract, essential oil and
ZER from Z. zerumbet have the potential to be used as
immunosuppressants to selectively inhibit the innate im-
mune responses consecutively at different stages.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12906-019-2748-5.
Additional file 1: Figure S1.
1
H NMR spectrum of zerumbone.
Additional file 2: Figure S2. HRESI-MS spectra of zerumbone.
Additional file 3: Figure S3. RP-HPLC chromatograms of (a) 80% etha-
nol extract of Zingiber zerumbet (b) zerumbone detected at 250 nm.
Abbreviations
CD: Cluster of differentiation; COX: Cyclooxygenase; DCM: Dichloromethane;
DMSO: Dimethylsulphoxide; DPPH: 2, 2-Diphenyl-1-Picrylhydrazyl; ESI-
MS: Electrospray ionization mass spectrometry; EtOAc: Ethyl acetate;
FACS: Fluorescence activated cell sorting; FBS: Fetal bovine serum;
Fc: Complement factor; FID: Flame ionization detector; FITC: Fluorescein
isothiocyanate; fMLP: formyl Methionine phenyl alanine; GC: Gas
Chromatography; HBSS: Hank’s balanced salt solution; HRESI-MS: High-
resolution electrospray ionization mass spectrometry; IgG: Immunoglobulin
G; LOD: Limit of detection; LOQ: Limit of quantification; LOX: Lipoxygenase;
LPS: Lipopolysaccharide; LTB
4
: Leukotriene B
4
; Mac-1: Macrophage-1 antigen;
MAPK: Mitogen-activated protein kinase; MPO: Myeloperoxidase; MS: Mass
spectrometry; MS: Mass spectrometry; NF-κB: Nuclear factor-kappa B;
NMR: Nuclear magnetic resonance; NSAID: Nonsteroidal Anti-inflammatory
Drugs; PBS: Phosphate buffer saline; PI3K: Phosphoinositide 3-kinase;
PMA: Phorbol myristate 13-acetate; PMNs: Polymorphonuclear neutrophils;
RI: Retention indices; ROS: Reactive oxygen species; RP-HPLC: Reversed phase
high performance liquid chromatography; RPMI: Roswell Park Memorial
Institute; RSD: Relative standard deviation; SOZ: Serum opsonized zymosan;
ZER: Zerumbone; ZZE: Zingiber zerumbet extract; ZZEO: Zingiber zerumbet
essential oil
Table 4 IC
50
values (μg/mL) of ROS inhibitory activity of tested samples on human whole blood and PMNs
Samples Zymosan PMA
Whole blood PMNs Whole blood PMNs
Z. zerumbet extract (ZZE) 6.49 ± 2.16 2.89 ± 0.98 10.87 ± 1.50 14.87 ± 2.70***
Z. zerumbet essential oil (ZZEO) 8.71 ± 1.37* 5.88 ± 1.70 11.63 ± 1.78 12.62 ± 0.87**
Zerumbone (ZER) –8.25 ± 0.73*
(37.77 ± 3.38)
6.83 ± 0.68
(31.25 ± 3.10)
3.79 ± 0.24
(17.36 ± 1.08)
Aspirin 2.63 ± 0.17
(14.57 ± 0.92)
1.95 ± 0.07
(10.82 ± 0.40)
7.65 ± 0.43
(42.46 ± 2.40)
2.94 ± 0.26
(16.30 ± 1.46)
Data are presented as (mean ± SEM, n = 3). Data were analyzed by one-way ANOVA followed by Dunnet’s post hoc test. IC
50
values in μM are shown in
parentheses. Significance of differences with respect to control: *p< 0.05, ** p< 0.01, ***p< 0.001
Akhtar et al. BMC Complementary and Alternative Medicine (2019) 19:331 Page 10 of 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Acknowledgements
The Ministry of Agriculture and Agro-based Industries, Malaysia is acknowl-
edged for the support under the NKEA Research Grant Scheme (NRGS).
Authors’contributions
NMYA performed the experiment, analyzed and interpreted the data, and
drafted the manuscript. IJ participated in the design, coordination of the
study, analysis and interpretation of data. He revised the manuscript and
approved the final version to be submitted for publication. LA and MAH
were involved in the analysis and interpretation of data. All authors read and
approved the final manuscript.
Funding
The study was funded by a grant from the Ministry of Agriculture and Agro-
based Industries, Malaysia (NKEA Research Grant Scheme, grant no.
NH1015D075). The funding body approved the design of the study, analysis,
and interpretation of data, and publication of the manuscript.
Availability of data and materials
Materials used and data collected in this study are available from the
corresponding author on reasonable request.
Ethics approval and consent to participate
The Human Ethical Committee of Universiti Kebangsaan Malaysia (Approval
no: UKM PPI/111/8/JEP-2017-335) approved the use of human blood in this
study. Volunteers who participated in this study provided written informed
consent for blood collection.
Consent for publication
Not applicable.
Competing interests
The authors have no conflict of interest, financial or otherwise.
Author details
1
Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti
Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia.
2
School of Pharmacy,
Faculty of Health and Medical Sciences, Taylor’s University, Lakeside Campus,
47500 Subang Jaya, Selangor, Malaysia.
3
Department of Pharmacy, Forman
Christian College (A Chartered University), Ferozeour Road, Lahore 54600,
Pakistan.
4
Department of Pharmacy, International Islamic University
Chittagong, Chittagong 4318, Bangladesh.
Received: 22 February 2019 Accepted: 7 November 2019
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