Environmental Science and Pollution Research (2021) 28:1131–1140

https://doi.org/10.1007/s11356-020-10559-9

RESEARCH ARTICLE

TLC bioautography–guided isolation of essential oil

components of cinnamon and clove and assessment of their
antimicrobial and antioxidant potential in combination
Shilpa Purkait1 & Abhishek Bhattacharya1 & Anwesa Bag1 & Rabi Ranjan Chattopadhyay1

Received: 15 May 2020 /Accepted: 17 August 2020 / Published online: 24 August 2020
# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
This study aimed to evaluate possible synergistic interactions on antimicrobial and antioxidant efficacy of clove and
cinnamon oil components incombination and characterization of compounds responsible for synergistic interactions using
TLC bioautography followed by checkerboard titration, isobologram analysis, and spectrometric characterization. Among
the combinations tested, cinnamaldehyde from cinnamon oil and eugenol from clove oil in combination showed a
synergistic antimicrobial interaction against foodborne microbes Listeria monocytogenes (fractional inhibitory
concentration index (FICI): 0.31), Salmonella typhimurium (FICI: 0.41), and Aspergillus niger (FICI: 0.48), and
synergistic antioxidant efficacy (combination index: 0.78) in in vitro model. Cinnamaldehyde/eugenol blend did not show
any cytotoxic effect (IC50 > 1000 μg/ml) in human normal keratinocyte cell line. The results provide evidence that the
cinnamaldehyde/eugenol blend may help in designing a more potent novel natural antimicrobial and antioxidant agent in
food and pharmaceutical industries.

Keywords Essential oil components . Antimicrobials . Antioxidants . Chemical characterization . Naturalfoodpreservatives

Introduction Rabi Ranjan Chattopadhyay

rabi@isical.ac.in
Foodborne disease caused by consumption of spoiled and 1

contaminated food is a major public health problem all Agricultural and Ecological Research Unit, Indian Statistical
Institute, 203, Barrackpore Trunk Road, Kolkata 700 108, India
over the world. Microbial contamination and oxidation of
Gould 2003). Therefore, there has been an increasing
food components especially lipids in food are responsible
interest among the researchers as well as food processors
for developing foodborne diseases (WHO 1984). Although
for the development of new types of effective and nontoxic
synthetic preservatives remain highly functional and cost
antimicrobial and antioxidantcompounds from natural
effective to overcome these problems, they have
sources aspromising alternatives to synthetic food
accumulated evidence that they could be toxic and
preservatives. In an effort to discover new lead
carcinogenic (Russell and
compounds, among the natural sources, many research
Responsible editor: Diane Purchase groups screen plant extracts to detect secondary
metabolites with relevant biological activities, because
* Shilpa Purkait plant secondary metabolites are rich sources of bioactive
shilpa.purkait.10@gmail.com
compounds and have many beneficial effects on human
Abhishek Bhattacharya health (Bhaskarachary et al. 2015; Tungmunnithum et al.
abhishekbhattacharya224@gmail.com 2018; Swallah et al. 2020). Various bioassay-guided
Anwesa Bag
isolation techniques are used by several workers for
anwesa.bag@gmail.com isolation of plant secondary metabolites from complex
plant extracts, of which the TLC bioautography technique
1132
is gaining much importance in recent years due to a
number of important considerations, viz., (i) this assay
allows a direct and rapid localization of bioactive
compounds in plant extracts; (ii) it is a fast, cheap, and
simple method for isolation of compounds from complex
plant extracts; (iii) it facilitates accurate identification of
chemical compounds in plant extracts; (iv) it is particularly
important to avoid the time-consuming isolation of inactive
compounds; and (v) this assay combines chromatographic
separation and in situ activity determination facilitating the
localization and target-directed isolation of active
constituents in a mixture (Hamburger and Cordell 1987;
Hostettmann et al. 2001; Shahverdi et al. 2007; Cheng and
Wu 2013). Our previous study revealed that cinnamon and
clove oils in combination exhibited synergistic
antimicrobial and antioxidant efficacy in combination
(Purkait et al. 2020). These synergistic antimicrobial and
antioxidant efficacy of cinnamon oil/clove oil combination
may help in designing a more potent novel natural
antimicrobial and antioxidant blend in the food and
pharmaceutical industries at sufficiently low
concentrations. These promising findings prompted us to
seek lead compounds responsible for synergistic
interactions to strengthen our findings. In the present study,
an attempt has therefore been made to seek lead
compounds responsible for synergistic antimicrobial and
antioxidant efficacy between cinnamon and clove oils in
combination by TLC bioautography–guided isolation
followed by checkerboard titration, isobologram analysis,
and spectrometric characterization techniques.
Materials and method
Essential oil extraction
Cinnamon (Cinnamomum verum; bark) and clove
(Syzygium aromaticm; bud) oils were extracted by using
the method described earlier (Purkait et al. 2020). Briefly,
the test spices were washed thoroughly in distilled water,
dried at 40 °C, powdered, and extracted by hydro-
distillation in Clevenger’s type apparatus. Anhydrous
sodium sulfate was used to maintain dryness and kept in
dark at 4 °C until used (yield: cinnamon oil (2.14%), clove
oil (2.85%)).
Microorganisms used
The foodborne microbes were selected based on their
relevance in the food industry. The microbial strains used
were pure reference standard foodborne pathogens Listeria
monocytogenes (MTCC 657) and Salmonella typhimurium
Environ Sci Pollut Res (2021) 28:1131–1140
(MTCC 3224) as indicator strains of Gram-positive and
Gram-negative foodborne bacteria respectively and
Aspergillus niger (ATCC 16404) as fungal strain. Bacterial
strains were procured from the Institute of Microbial
Technology, Chandigarh, India, and fungal strain was
procured from National Chemical Laboratory, Pune, India.
They were maintained following the standard CLSI
guidelines for bacteria (CLSI 2005) and CLSI M38-A2
guidelines for molds (CLSI 2008).
Inoculum size standardization
For standardization of bacterial inoculum size, L.
monocytogenes was incubated at 30 °C and
S. typhimurium at 37 °C for 3–6 h to get 0.5 McFarland
unit of turbidity. The size of the inoculum was adjusted to
5 × 105 CFU/ml (CLSI 2005). For standardization of fungal
inoculum size, at first, A. niger inoculum suspension was
made by inoculating sporulated fungus with a loop in 10
ml of sterile water from fresh, matured (3 to 4 days old)
culture of A. niger grown on potato dextrose agar slant at
35 °C. The fungal suspension was then filtered to remove
hyphae. The filtered suspension of A. niger was vigorously
vortexed and adjusted to 5 × 105 CFU/ml (CLSI 2008).
TLC bioautography for screening of antimicrobial
components
Analytical TLC for Rf value determination
Analytical TLC was done on TLC plates (5 × 10 cm, 0.25-
mm thickness, Silica gel G 60 F254, Merck, Darmstadt,
Germany) for the determination of R f of separated
components of cinnamon and clove oils using the
procedure of Suleimana et al. (2010). Briefly, the TLC
plate was first preconditioned at 120 °C for 1 h, then 5 μl
(10 mg/ml) of oil was loaded on the TLC plate with
capillary pipettes and air dried. The plate was developed
using a solvent mixture of toluene:ethyl acetate (95:5 v/v)
in a presaturated glass chamber for 3–4 h. After
developing, the plate was dried and heated for 5 min at 110
°C for spot visualization of separated components. R f
values of separated components in the plate were
determined. For agar overlay bioautography, analytical
TLC plates without spraying reagent were also prepared
simultaneously to avoid possible interference of visualizing
reagent on microbiocidal activity of test samples in
bioautograms (Suleimana et al. 2010).
Environ Sci Pollut Res (2021) 28:1131–1140
Agar overlay bioautography
For detection of antimicrobial components present in oils,
agar overlay bioautography was used. For this, developed
analytical TLC plates that received no spraying reagent
were kept in sterile petri plates, and then 100 μl inoculum
of each of test microorganisms (5 × 10 5 CFU/ml) for every
10 ml of melted nutrient agar (for bacteria) or potato
dextrose agar (for fungi) was given over the plates
separately. After solidification, plates were incubated at 30
°C for L. monocytogenes and 37 °C for S. typhimurium for
24 h and at 35 °C for fungal strain (A. niger) for 48 h. Then
p-iodonitrotetrazolium violet (INT) (0.2 mg/ml) solution
was sprayed on developed bioautograms. Antibacterial and
antifungal components were identified as clear inhibition
zones (white spots) against a pink-colored background that
indicated microbial growth (Shahverdi et al. 2007). Rf
values of inhibition zones (white spots) in the agar overlay
plates were determined from corresponding Rf values in the
analytical plate obtained by spraying with p-anisaldehyde-
sulfuric acid reagent.
Preparative TLC
Antimicrobial components present in oils were isolated by
using the preparative TLC procedure. For this, streak of
oils were loaded on preparative TLC glass plates (20 × 20
cm; 1mm thickness) (Sigma-Aldrich, USA) and air dried.
After air drying, plates were developed using toluene:ethyl
acetate (95:5 v/v) solvent mixture. Then, 100 μl inoculum
of test bacterial or fungal strains (5 × 10 5 CFU/ml) for
every 10 ml of melted nutrient agar (for bacteria) or potato
dextrose agar (for fungi) was given over the plates. The
plates were incubated and sprayed with INT solution
following the same procedure as mentioned in agar overlay
bioautography. The inhibition bands of antibacterial or
antifungal components were carefully scratched off and
dissolved in 80% methanol. The scratched materials in
methanol were centrifuged at 10,000×g for 15 min, and
supernatants were collected, filtered through 0.22-μm
filter, and dried in vacuo. The dried antimicrobial
components with different Rf values were collected and
stored at 4 °C.
TLC bioautography for detection of antioxidant
components
Detection of antioxidant components present in oils was
performed by TLC bioautography following the procedure
described byLihuaet al. (2009).Briefly, atfirst,a TLC plate
(5× 10 cm, 0.25-mm thickness, Silica gel G 60 F254,
Merck, Darmstadt, Germany) was preconditioned at 120
1133
°C for 1 h. Then 5 μl (10 mg/ml) of test oil was loaded to
the TLC plate with capillary pipette, dried, and developed
using toluene:ethyl acetate (95:5 v/v) solvent mixture in a
presaturated glass chamber for 3–4 h at room temperature.
After development, the plate was placed in sterile petri
dish, sprayed with DPPH solution (0.02% w/v in methanol)
and heated for 5 min at 110 °C. Development of bright
yellow to pink color for confirmation as antioxidant
components was observed (Hafid et al. 2014). Rf values of
spots of separated antioxidant components in the plate
were determined.
Preparative TLC
Antioxidant components of oils were isolated by using the
preparative TLC procedure using a preparative TLC glass
plate (20 × 20 cm, 1-mm thickness) (Sigma-Aldrich,
USA). Briefly, a streak of oil was loaded on the plate and
air dried. The plate was developed in a pre-saturated glass
chamber using a solvent mixture of toluene:ethyl acetate
(95:5 v/v) for 3–4 h. The developed bioautograms were
sprayed with 0.02% (w/v) DPPH solution in methanol and
heated for 5 min at 110 °C. The bands showing antioxidant
activity were carefully scratched off and dissolved in 80%
methanol. The scratched antioxidant materials in methanol
were then centrifuged at 10,000×g for 15 min; supernatants
were collected, filtered through 0.22-μm filter, and dried in
vacuo. The dried antioxidant components with different R f
values were collected and stored at 4 °C until used.
Among the isolated components obtained by TLC
screening, only components that demonstrated both
antimicrobial and antioxidant efficacy in bioautograms
were considered “active essential oil components
(AEOCs)” and used for combined antimicrobial and
antioxidant activity study.
Evaluation of combined antimicrobial potential
Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) of AEOCs was
determined at their individual effect following the CLSI
(2005) guidelines. Briefly, 100 μl of reconstituted solution
of AEOCs with respective broth at various concentrations
(1.56 to 200 μg/ml) was added in wells of microtiter plates
containing 90 μl of selective broth. Then, 10 μl of
inoculum (5 × 105 CFU/ml) was added to each well.
Negative control wells received respective broth instead of
AEOCs. Plates were then incubated at 30 °C for L.
monocytogenes and 37 °C for S. typhimurium for 24 h.
Then, 40 μl (0.4 mg/ml) of INT solution was added to
1134
wells and further incubated for 6 h. The lowest dilution of
AEOCs with no color change was considered MIC. Each
experiment was repeated thrice. Determination of
minimum fungicidal concentration
Minimum fungicidal concentrations (MFCs) of AEOCs at
their individual effect against fungal strain A. niger were
determined following the CLSI M38-A2 guidelines (CLSI
2008). Briefly, 100 μl of reconstituted solution of AEOCs
with RPMI-1640 medium at various concentrations (1.56
to 200 μg/ml) was added in wells of the microtiter plate.
Then, 100 μl of A. niger inoculum (5 × 10 5 CFU/ml) was
added in each well and incubated for 48 h at 35 °C. Wells
containing A. niger inoculum and RPMI 1640 instead of
AEOCs served as negative control. After incubation, 40 μl
of INT solution (0.4 mg/ml) was added and further
incubated for 6 h. MFC was defined asthe lowest
concentration that showednovisible growth. Each
experiment was repeated thrice.
Determination of fractional inhibitory concentration index
For the determination of fractional inhibitory concentration
index (FICI) values of oils, at first, MIC and MFC of
AEOCs in combination (1:1) were determined following
the guidelines of CLSI (2005) for bacteria and CLSI
(2008) for fungi. For the determination of MIC in
combination against bacterial strains, 10 μl of bacterial
inoculum (5 × 105 CFU/ml) was added in wells of the
microtiter plate containing 90 μl of respective broth. Then,
AEOCs (100 μl) in combination (1:1) at various
concentrations(1/32 × MIC to4 × MIC)wereadded in wells
and incubated for 24 h at 30 °C for L. monocytogenes and
at 37 °C for S. typhimurium. For the determination of
MFC in combination against fungal strain, 100 μl of fungal
suspension (5 × 105 CFU/ml) and 100 μl of AEOCs in
combination (1:1 v/v) at various concentrations (1/32 ×
MFC to 4 × MFC) were added in wells of the microtiter
plate and incubated for 48 h at 35 °C. The procedures for
combined MIC and MFC determination are same as for
individual MIC and MFC determination. FICI was
determined as follows: FICI = (MIC or MFC of AEA in
the presence of AEB/MIC or MFC of AEA alone) + (MIC
or MFC of AEB in the presence of AEA/MIC or MFC of
AEB alone), where AEA and AEB are two different
AEOCs. The antimicrobial interactions between two
AEOCs were interpreted as follows: synergy (FICI = 0.5),
additive (0.5 < FICI = 4), and antagonistic (FICI > 4)
(Leclercq et al. 1991).
Environ Sci Pollut Res (2021) 28:1131–1140
Evaluation of in vitro antioxidant potential in combination
DPPH radical scavenging efficacy
The DPPH (1, 1-diphenyl 2-picrylhydrazyl) radical
scavenging activity of AEOCs alone and in combination
was evaluated following the method of Wang et al. (1998).
Briefly, AEOCs (100 μl) alone and in combination (1:1) at
varying concentrations (1.56 to 200 μg/ml) were added in
tubes containing 3.9 ml (0.1 mM) of DPPH in methanol. In
the control tube, methanol was used and kept in dark at
room temperature for 30 min. The absorbance was
measured at 517 nm. The free radical scavenging activity
was calculated using the following equation.
ð Þ% Free radical scavenging ¼ Acontrol−Asample=Acontrol 100
where Asample denotes absorbance of DPPH treated with
AEOCs and Acontrol denotes absorbance of DPPH treated
with methanol.
Determination of combination index
The combination index was determined on the basis of IC 50
values of AEOCs using isobologram analysis (Chou et al.
1994) as follows.
CI ¼ ð ÞD 1=ðDxÞ1 þð ÞD 2=ðDxÞ2
where (D)1 and (D)2 are IC50 values of two AEOCs in
combination, and (Dx)1 and (Dx)2 are IC50 values of two
AEOCs individually. Based on combination index (CI)
values, the type of antioxidant interaction was interpreted
as CI < 1: synergistic, CI = 1: additive, and CI > 1:
antagonistic (Chou et al. 1994).
Cytotoxicity study
Cell culture
The cytotoxicity study of AEOCs that exhibited synergistic
interactions in combination was evaluated using human
normal keratinocyte cell line (HaCaT cells). The
immortalized human keratinocyte cell line was obtained
from the Human Genetics Unit, Indian Statistical Institute,
Kolkata, India. Cells were maintained at 37 °C in a
humidified atmosphere containing 5% CO2 in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with
10% FBS, 2 mM glutamine, and 1%
penicillin/streptomycin (growth medium). Cytotoxicity
study was performed in 96-well plates using WST-1-based
colorimetric assay. Briefly, 100 μl (1 × 104 cells/well) of
Environ Sci Pollut Res (2021) 28:1131–1140
cell suspensions was seeded for 24 h in a 96well culture
plate. The cells were then exposed to different
concentrations (15.62, 31.25, 62.5, 125, 250, 500, 1000
μg/ml) of selective AEOCs in combination (1:1) and
incubated at 37 °C for 72 h in a humidified atmosphere
containing 5% CO2. After incubation, 10 μl WST-1 reagent
(Roche, Mannheim, Germany) was added. Plates were
incubated for further 24 h in the previously mentioned
conditions, and absorbance was measured at 450 nm. The
positive control tube received 1% Triton X-100, and
culture medium was used as negative control. Percentage
of cell viability was determined as follows.
Cell viability (%) = (absorbance of treated sample/
absorbance of negative control) × 100
Spectrometric characterization
Among the AEOCs tested, only one component of
cinnamon oil (Rf: 0.75) and one component of clove oil
(Rf: 0.42) showed synergistic antimicrobial and antioxidant
activities in combination. These two components were then
subjected to spectrometric (UV/Vis, Fourier-transform
infrared (FT-IR), and gas chromatography high-resolution
mass spectrometry (GC-HRMS)) analysis for chemical
characterization of compounds.
UV/Vis absorption spectra
UV/Vis absorption spectra of AEOCs (R f: 0.75) and clove
(Rf: 0.42) were recorded using a Perkin Elmer Lamda15
UV-Vis spectrometer at 200–850 nm.
FT-IR analysis
The FT-IR spectroscopic analysis of AEOCs of interest
was done by using the Fourier-transform infrared
instrument (make and model: Perin Elmer Spectrum 1
spectrometer) in the scan range 450–4000 cm−1.
GC-HRMS analysis
1135
by comparison of mass spectra
ofpurecompoundwithliterature data aswell asfrom the
NIST 02 MS library of pure compounds.
Statistical analysis
Statistical analysis of data was performed using the SPSS
software: version 18.0. A one-way ANOVA (analysis of
variance) followed by Tukey’s range test was used for
analysis of data with the level of significance set at P <
0.05.
Results and discussion
Table 1 shows Rf values of bioactive essential oil
components of cinnamon and clove. A total of 11
components from oils weredetected in TLC bioautography,
of which onlythree components of clove oil (Rf: 0.42, 0.73,
and 0.82) and two components of cinnamon oil (R f: 0.24
and 0.75) showed both antimicrobial and antioxidant
activities in bioautograms. These five selected AEOCs
were used for in vitro antimicrobial and antioxidant
efficacy evaluation study.
Table 2 shows the MIC and MFC values of five selected
active components alone and in combination against the
studied microbes. Among the possible combinations tested,
MIC and MFC values of combination between the clove oil
component (Rf: 0.42) and the cinnamon oil component (Rf:
0.75) were significantly (P < 0.05) lowered compared with
the MIC and MFC values of their most active single
component, whereas other tested possible combinations
failed to do so.
Next, the typeofantimicrobial
interactions(additive,synergistic, or antagonistic) in
combinations between selected five essential oil
components was tested (Table 3). Based on FICI values,
the clove oil

The EOCs of interest (Rf: 0.75) and clove (Rf: 0.42) were
subjected to GC-HRMS analysis for determination of
AntioxidantAntioxidant

molecular mass of compounds using an Agilent 7890 GC

model system mass spectrometer (Jeol, model: Accu TOF
oil

GCF). Pure helium (He) was used as carrier gas with a

0.42*0.24*
0.73*0.75*

flow rate of 1.0 ml/min. The column used was HP-5MS.

0.230.16
CinnamonoilCloveoilCinnamon

The injector temperature was 200 °C, and the detector

0.82*-

temperature was 280 °C. Mass spectral scan range was set
at 10–2000 amu, and mass resolution was 6000. The
identification of the individual components was performed
A.niger16404)

0.24*
0.75*
ATCC
yscreening

-
-
(
 S.typhim
32
nessentialoilsobtainedbyTLC bioauto

0.24*
0.75*
MTCC
CinnamonoilCinnamonoil

andantioxidantefficacyinTLCbioautographyscreening
-
-
1136 Environ Sci Pollut Res (2021) 28:1131–1140

L.monocytogenes

(
component (Rf: 0.42) and cinnamon oil component (Rf: (FICI: 0.48). Other tested combinations showed an additive
0.75) in combination exhibited synergistic antimicrobial antimicrobial effect against all the studied microbes with

657)
efficacy against both the studied bacterial strains L. FICI ranged from 1.71 to 2.03 (Table 3).

MTCC
0.24*

0.75*
monocytogenes (FICI: 0.31) and S. typhimurium (FICI:

0.43
0.38
0.41) and fungal strain A. niger

(
Table 2 Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of active components of clove and

mponentsofcloveandcinnamo
cinnamon oils at their individual and combined effects

16404)
Test essential oils R f value of active components MIC ( μ g/ml) MFC ( μ g/ml)

Cloveoil

A.niger

0.42*
0.73*
0.82*
ATCC
L. monocytogenes S. typhimurium A. niger

-
(MTCC 657) (MTCC 3224) (ATCC 16404)

sthatshowedbothantimicrobial
(
Clove oil components (individual
effect)

S.typhimurium
3224)
Clove oil 0.42 70.74 ± 2.16 85.43 ± 3.54 68.73 ± 2.81

Rf valuesofantimicrobialandantioxidantco
Clove oil 0.73 92.65 ± 4.67 106.53 ± 3.28 108.63 ± 3.74

Cloveoil

MTCC
0.42*
0.82*
Clove oil 0.82 79.86 ± 3.75 89.28 ± 3.52 83.64 ± 3.76
Cinnamon oil components (individual effect)

-
-
Cinnamon oil 0.24 92.72 ± 3.83 102.42 ± 3.27 98.82 ± 4.52

(
L.monocytogenes
Cinnamon oil 0.75 75.18 ± 2.16 114.63 ± 3.44 77.43 ± 2.64

*Activeessentialoilcomponent
Clove oil and Cinnamon oil components (combined effect)

657)
Clove oil + cinnamon oil 0.42 + 0.24 74.64 ± 2.17 (Rf 0.42) 89.63 ± 4.27 (Rf 0.42) 72.54 ± 3.78 (Rf 0.42)

MTCC
( 0.42*
0.73*
0.82*
0.14
94.73 ± 2.83 (Rf 0.24) 97.25 ± 4.83 (Rf 0.24) 91.62 ± 4.77 (Rf 0.24)

EssentialoilCloveoil

Microorganisms
Clove oil + cinnamon oil 0.42 + 0.75 10.37 ± 1.69 (Rf 0.42)* 17.42 ± 2.32 (Rf 0.42)* 14.84 ± 1.32 (Rf 0.42)*
12.86 ± 1.45 (Rf 0.75)* Table1 24.65 ± 3.43 (Rf 0.75)* 21.57 ± 2.18 (Rf 0.75)*

Clove oil + cinnamon oil 0.73 + 0.24 89.47 ± 3.46 (Rf 0.73) 103.81 ± 3.32 (Rf 0.73) 97.88 ± 3.28 (Rf 0.73)
93.22 ± 3.54 (Rf 0.24) 110.72 ± 4.43 (Rf 0.24) 96.76 ± 3.79 (Rf 0.24)

Rf
Clove oil + cinnamon oil 0.73 + 0.75 80.44 ± 3.37 (Rf 0.73) 104.78 ± 4.54 (Rf 0.73) 102.65 ± 4.82 (Rf 0.73)
76.77 ± 3.92 (Rf 0.75) 112.93 ± 4.72 (Rf 0.75) 80.53 ± 4.19 (Rf 0.75)

Clove oil + cinnamon oil 0.82 + 0.24 75.43 ± 3.26 (Rf 0.82) 92.76 ± 3.26 (Rf 0.82) 86.54 ± 3.45 (Rf 0.82)
72.14 ± 4.10 (Rf 0.24) 98.97 ± 4.22 (Rf 0.24) 95.69 ± 4.80 (Rf 0.24)

Clove oil + cinnamon oil 0.82 + 0.75 73.65 ± 3.84 (Rf 0.82) 90.53 ± 3.21 (Rf 0.82) 81.29 ± 3.83 (Rf 0.82)
78.42 ± 4.77 (Rf 0.75) 109.67 ± 5.41 (Rf 0.75) 75.44 ± 3.65 (Rf 0.75)

Clove oil + clove oil
Clove oil + clove oil
0.42 + 0.73 69.67 ± 3.73 (Rf: 0.42) 82.47 ± 3.22 (Rf: 0.42) 71.46 ± 3.90 (Rf: 0.42)
90.84 ± 4.82 (Rf:0.73) 103.94 ± 3.92 (Rf: 0.73) 109.33 ± 4.78 (Rf:
0.73)
0.42 + 0.82 69.68 ± 3.82 (Rf: 0.42) 83.29 ± 3.52 (Rf: 0.42) 70.33 ± 3.92 (Rf: 0.42)
78.46 ± 3.78 (Rf: 0.82) 90.73 ± 3.80 (Rf: 0.82) 79.65 ± 4.84 (Rf: 0.82)

Clove oil + clove oil 0.73 + 0.82 87.32 ± 4.61 (Rf: 0.73) 104.59 ± 3.89 (Rf: 0.73) 105.43 ± 4.90 (Rf:
0.73)
80.70 ± 3.88 (Rf: 0.82) 90.14 ± 3.68 (Rf: 0.82) 81.47 ± 4.21 (Rf: 0.82)

Cinnamon oil + cinnamon oil 0.24 + 0.75 85.77 ± 4.18 (Rf: 0.24) 103.27 ± 3.26 (Rf: 0.24) 95.34 ± 3.72 (Rf: 0.24)
77.87 ± 3.84 (Rf: 0.75) 112.67 ± 4.38 (Rf: 0.75) 78.71 ± 3.77 (Rf: 0.75)

Values are mean ± SD The literature reveals that olive oil, walnuts, and marine
*P < 0.05 compared with their individual effect fish are a heart-healthy choice, but concern is rising that if
not stored and used properly, these foods and oils actually
could be harmful for our health due to auto-oxidation of
Environ Sci Pollut Res (2021) 28:1131–1140 1137
fats and oils in food. For this reason, in the food industry,
antioxidants are also used for retarding oxidation of food
components especially fatty acids in food (Yadav et al.
2016). Therefore, in the present study, we evaluated
antioxidant potential of five selected active essential oil
components of clove and cinnamon in combination, taking
the advantages of their possible synergistic interaction.
Among the combinations tested, only one combination
between the clove oil component (Rf: 0.42) and the
cinnamon oil component (Rf: 0.75) showed synergistic
antioxidant efficacy (CI = 0.72), whereas other tested
combinations showed an additive antioxidant effect (CI =
1) (Table 4).
Now, essential oil components of clove (Rf: 0.42) and
cinnamon (Rf: 0.75) that exhibited synergistic antimicrobial
and antioxidant efficacy in combination were evaluated for
their cytotoxic potential, if any using Triton X-100 (1%) as
positive control. The test essential oil components in
combination did not show any cytotoxic potential up to
1000-μg/ml concentration in normal cell line (IC 50 > 1000
μg/ml) (Fig. 1). Literature reveals that recommended doses
of plant extracts for an in vitro activity study should be less
than 100 μg/ml, and for pure bioactive compounds, it is
below 25 μM (Cos et al. 2006; Soh and Benoit-Vical
2007). To estimate the potential of bioactive compounds or
plant extracts to inhibit antimicrobial
1138 Environ Sci Pollut Res (2021) 28:1131–1140
Table 3 Fractional inhibitory concentration index (FICI) of active essential oil components of clove and cinnamon in combination
Test essential oils Rf value of
Foodborne microbes
active
components L. monocytogenes S. typhimurium A. niger
(MTCC 657) (MTCC 3224) (ATCC
16404)
FIC FIC
FICI Remarks FICI Remarks FIC FICI Remarks
Clove oil +
cinnamon oil 0.42 + 0.24 1.05 2.07 ADD 1.04 1.99 ADD 1.05 1.97 ADD
1.02 0.95 0.92

Clove oil + 0.42 + 0.75 0.14 0.31 S 0.20 0.41 S 0.21 0.48 S
cinnamon oil
0.17 0.21 0.27

Clove oil + 0.73 + 0.24 0.96 1.96 ADD 0.97 2.05 ADD 0.90 1.87 ADD
cinnamon oil
1.00 1.08 0.97

Clove oil + 0.73 + 0.75 0.86 1.88 ADD 0.98 1.96 ADD 0.94 1.98 ADD
cinnamon oil
1.02 0.98 1.04

Clove oil + 0.82 + 0.24 0.94 1.71 ADD 1.03 1.99 ADD 1.03 1.99 ADD
cinnamon oil
0.77 0.96 0.96

Clove oil + 0.82 + 0.75 0.92 1.96 ADD 1.01 1.96 ADD 0.97 1.94 ADD
cinnamon oil
1.04 0.95 0.97

Clove oil + clove 0.42 + 0.73 0.98 1.96 ADD 0.96 1.83 ADD 1.03 2.03 ADD
oil
0.98 0.97 1.00

Clove oil + clove 0.42 + 0.82 0.98 1.96 ADD 0.97 1.98 ADD 1.02 1.97 ADD
oil
0.98 1.01 0.95

Clove oil + clove 0.73 + 0.82 0.94 1.94 ADD 0.98 1.98 ADD 0.97 1.94 ADD
oil
1.00 1.00 0.97

Cinnamon oil + 0.24 + 0.75 0.92 1.95 ADD 1.00 1.98 ADD 0.96 1.97 ADD
cinnamon oil
1.03 0.98 1.01

Synergistic: FICI ≤ 0.5, additive: 0.5 > FICI ≤ 4, antagonistic: FICI > 4
S, synergistic; ADD, additive
activity without toxicity, the selectivity index (SI) was
introduced.The SI is the ratio between cytotoxic
activity(IC50) and antimicrobial activity (MIC 50). Low SI
indicates that the antimicrobial activity is probably due to
cytotoxicity rather than
activity against the test microbes. In contrast, a high SI (SI
≥ 10) offers a potentially safer therapy (Valdes et al. 2010).
Environ Sci Pollut Res (2021) 28:1131–1140 1139
According to the American National Cancer Institute considered
(NCI) plant screening program, a crude extract is generally
Table 4Antioxidant combination index (CI) values of active essential oil components of cinnamon and clove in combination
Test essential oils Rf value of active IC50 (μg/ml) CI1 = (D)1/(Dx)1 CI2 = (D)2/(Dx)2 CI = CI1 + CI2 Remarks
components
Clove oil 0.42 21.48 ± 1.04 - - - -
Clove oil 0.73 32.66 ± 2.37 - - - -
Clove oil 0.82 26.78 ± 2.81 - - - -
Cinnamon oil 0.24 34.07 ± 2.63 - - - -
Cinnamon oil 0.75 23.95 ± 1.75 - - - -
Clove oil + cinnamon oil 0.42 + 0.24 13.33 ± 1.98 0.61 0.39 1.00 ADD
Clove oil + cinnamon oil 0.42 + 0.75 8.25 ± 0.89 0.38 0.34 0.72 S
Clove oil + cinnamon oil 0.73 + 0.24 16.66 ± 1.56 0.51 0.49 1.00 ADD
Clove oil + cinnamon oil 0.73 + 0.75 13.69 ± 2.01 0.42 0.58 1.00 ADD
Clove oil + cinnamon oil 0.82 + 0.24 15.15 ± 2.66 0.56 0.44 1.00 ADD
Clove oil + cinnamon oil 0.82 + 0.75 12.65 ± 1.78 0.47 0.53 1.00 ADD
CI < 1 (synergistic), CI = 1 (additive)
IC50: the half maximal inhibitory concentration of
active essential oil components 120
Fig. 1 Cytotoxic potential of essential oil
components of cinnamon (R f: 0.75) and clove (Rf: 100
0.42) in combination. *No significant (P < 0.05)
difference in cell viability up to 1000-μg/ml 80
Cell viability (%)

concentration when compared with negative

control but significantly (P < 0.05) different when 60
compared with positive control Triton X-100
(1%) 40
to have an in vitro cytotoxic activity with
an IC50 value ≤ 30 μg/ml. However, a 20
crude extract with IC50 < 20 μg/ml is
0
considered to be highly cytotoxic. The
IC50 value of cytotoxic pure compounds
will be lower than that specified by NCI,
USA, for crude plant extracts Conc (µg/ml)
(Vijayarathna and Sasidharan 2012;
Sriwiriyajan et al. 2014). In our present investigation, IC50
of test bioactive compounds in combination against
keratinocyte cell line was found to be > 1000 μg/ml. On
the other hand, MIC and MFC values of test bioactive
compounds (that showed synergistic antimicrobial
interactions in combination) against the studied microbes
were ranged from 10.37 ± 1.69 to 24.65 ±
3.43 μg/ml. Therefore, SI values of these two bioactive
compounds in combination are too high compared with a
safer range (SI ≥ 10). This findings suggest that test
essential oil components in combination can generally be
considered safe at a recommended dosage level
(Sriwiriyajan et al. 2014).
1140 Environ Sci Pollut Res (2021) 28:1131–1140
Next, the cinnamon oil component (Rf: 0.75) and the reported synergistic antibacterial, antifungal, and
clove oil component (Rf: 0.42) that were found to have antioxidant activities of cinnamaldehyde/eugenol in
synergistic combination.
antimicrobialandantioxidantefficacyincombinationweresub Collectively, better antibacterial, antifungal, and
jectedto spectrometric (UV/Vis, FT-IR, GC-HRMS) antioxidant activities of cinnamaldehyde/eugenol
analyses for chemical characterization of compounds. The combination observed in the present investigation have a
obtained spectral data of cinnamon and clove oil significant impact on the food preservation system as it
components are shown in Table 5. The collective will help in better preservationoffoods due tobroader
spectrometricanalysesdata revealed thatthecinnamon oil spectrum ofantimicrobial and antioxidant efficacy at
component (Rf: 0.75) and the clove oil component (R f: sufficiently low concentrations. The combination of these
Table 5 Spectrometric analyses data of essential oil components of cinnamon (R f: 0.75) and clove (Rf: 0.42)
Parameters Clove oil component (Rf: 0.42) Cinnamon oil component (Rf: 0.75)

UVλmax (nm) 280 (assigned to the aromatic O–H group)
−
3513.96 (alcohol O–H stretching); 3074.31
FT-IR (cm 1)
(SP2C–H stretching); 3005.71–2842.54
(SP3 C–H stretching); 1848.74 (C=O
stretching); 1637.99–1608.74 (alkene
C=C stretching); 1513.83–1458.91
(aromatic C=C stretching); 1432.86–
1367.86 (aromatic C=C stretching);
1268.9–1206.26 (phenol C–O); 1034.08–
648.48 (alkene SP2 C–H bending)
GC-HRMS Retention time (min) 22.53
Mass of parent ion (m/z) 164.0983
285 (assigned to the –CHO group)
3335.81 (SP C–H stretching);
3060.73–3031.06 (SP2 C–H stretching);
2928.48 (SP3 C–H stretching);
2817.06–2743.60 (aldehyde C–H)
1971.83–1493.14 (aldehyde C=O and
aldehyde C–H); 1450.85 (aromatic
C=O stretching); 1392.5–1296.72
(SP3 C–H bending); 1247.54–1125.33
(phenol C–O); 1073.01–635.38
(alkene SP2
C–H bending)
18.90
131.0539

Mass of fragment ions (m/z) 149.0730, 131.0597, 121.0742, 103.0592, 103.0544, 77.0322, 51.0051
77.0356, 55.0051
Identified compound 4-Allyl-2-methoxyphenol (eugenol) (2E)-3-Phenylprop-2-enal
(trans-cinnamaldehyde)
Molecular formula C8H10O C9H8O
Molecular structure NIST MS search, v. 2.0 [14, 15]
References
NIST MS search, v. 2.0 [14, 15]
0.42) two essential oil components will also be helpful in
closely correspond with that of the authentic sample of reducing concentration of individual components, thereby
(2E)-3phenylprop-2-enal (trans-cinnamaldehyde) and 4- minimizing the undesirable impact on sensory properties of
allyl-2methoxyphenol (eugenol) respectively as evidenced food. To our knowledge, this is the first time that
from the NIST MS search (v.2.0) and available literature synergistic antimicrobial and antioxidant efficacy of
data (Adams 2007; Berger and Sicker 2009). essential oil components in combination has been reported
Thus, the bioactive compounds of cinnamon and clove by us.
oils responsible for synergistic antimicrobial and
antioxidant activities are (2E)-3-phenylprop-2-enal (trans- Acknowledgments The authors are thankful to the Dean of Studies,
cinnamaldehyde) and 4-allyl-2-methoxyphenol (eugenol) Indian Statistical Institute, Kolkata, India, for providing research
fellowship to the corresponding author for pursuing this research
respectively (Table 5). Literature reveals that work. The authors also wish to acknowledge the Heads, SAIF, Indian
cinnamaldehyde/eugenol combination exhibited a Institute of Technology, Madras, India, and Central Drug Research
synergistic antimicrobial activity against Escherichia coli Institute, Lucknow, India, for providing laboratory facilities to carry
and wood decay fungi Laetiporus sulphurous (Pei et al. out spectrometric analysis of samples.
2009; Yen and Chang 2008). Besides, the antioxidant
effect of cinnamaldehyde and eugenol at their individual
effect has alsobeendocumented(Ozcanand Arslan 2011;
Sharma et al. 2017). In the present investigation, we
Environ Sci Pollut Res (2021) 28:1131–1140
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical statement This article does not contain any studies with
human participants or animals performed by any of the authors.
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