Industrial Crops & Products 138 (2019) 111578

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Industrial Crops & Products

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Extraction of betel leaves (Piper betle L.) essential oil and its bio-actives T
identification: Process optimization, GC-MS analysis and anti-microbial
activity
⁎
Mitali Madhumitaa, , Proshanta Guhaa, Ahindra Nagb
a
Department of Agricultural and Food Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India
b
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India

A R T I C LE I N FO A B S T R A C T

Keywords: Betel leaf (Piper betle L.) var. Bangla is an essential oil (EO) rich plant belongs to Piperaceace family used as
Fresh and cured betel leaves traditional herbal medicine. The extraction of EO from fresh and cured betel leaves is of high interest for in-
Essential oil dustrial application. Thus, the present study was aimed to optimize the EO extraction and its bio-chemical
GC–MS analysis characterisation. Box-Behnken Design coupled with response surface methodology was employed to optimize the
Antibacterial activity
independent variables such as liquid to solid ratio (30:1-50:1 mL/g), extraction time (5–7 h), and particle size
SEM
(0–20 mesh). Quadratic polynomial models were found to be highly significant (p < 0.05) obtaining fresh and
cured betel leaf EO with a yield of 0.18% ± 0.01% and 0.22% ± 0.02% at optimal condition: 40:1 mL/g, 6 h,
and 10 mesh. Gas chromatography mass spectrometry (GC–MS) analysis revealed thirty-three and thirty volatile
compounds, representing 98.41% and 97.34% of the optimized fresh and cured leaf EO, respectively which have
a varied range of biological activities and industrial applications. Eugenol, estragole, linalool, α-copaene, an-
ethole, chavicol, and caryophyllene were found to be highly abundant in both EO with different percentages.
Antibacterial activity was evaluated against Mycobacterium smegmatis, Staphylococcus aureus and Pseudomonas
aeruginosa and the results demonstrated that cured leaf EO showed significantly higher antimicrobial activity
against M. smegmatis than fresh leaf EO. Moreover, morphological study was carried out to investigate the
extraction mechanism of fresh and cured betel leaves with the help of scanning electron microscopy (SEM).

1. Introduction
Betel leaves (Piper betel L.), is a perennial traditional herbal plant
belonging to Piperaceae family and originated in Malaysia. Over 700
species of Piper betel were discovered in both Northern and Southern
hemispheres of the world and widely grown in most of the countries
such as India, Sri Lanka, Malaysia, Indonesia, Philippines, and other
Southeast Asian and East African countries (Umar et al., 2018). This
plant is popularly known as Paan and widely cultivated as well as
consumed in all over India (Guha, 2006). It is an evergreen aquatic root
climbing vine with dorsiventral heart shaped leaves which are con-
sumed regularly by a large number of the Asians. The leaves having
high pungent palatable taste causes irritation in mouth to some con-
sumers. Therefore, sometimes this pungency is removed by a special
smoke treatment called curing. Cured betel leaves were prepared by
local farmers of West Bengal within a closed chamber. In that chamber,
fresh green betel leaves were treated with smoke at a temperature of
50ᵒC for 6 h and then cooled at room temperature for 12 h. The green
betel leaves were converted to yellow leaves (i.e. called cured leaves)
after incubating for 15–20 days (Guha, 2007). The cured leaves produce
better taste, colour (light yellow to white) and other attractive orga-
noleptic properties besides stimulating and refreshing effects. The leaf
has got a good nutritive value particularly due to its high mineral
contents (mainly calcium), vitamins and bioactive compounds like
phenolic, flavonoid, essential oil (EO) etc. Betel leaves contain a vola-
tile EO contributing most of its medicinal, organoleptic, and other de-
sirable properties. Essential oil is a mixture of a large number of volatile
compounds (secondary metabolites) having complex composition with

Abbreviations: EO, essential oil; HD, hydro distillation; BBD, Box-Behenken Design; RSM, response surface methodology; ANOVA, analysis of variance; FLEO, fresh
leaf essential oil; CLEO, cured leaf essential oil; GC–MS, gas chromatography–mass spectrometry; HPLC, high-performance liquid chromatography; LC–MS, liquid
chromatography with mass spectrometry; NMR, nuclear magnetic resonance spectrometry; FT-IR, Fourier transform infrared; d.b., dry basis; SEM, scanning electron
microscopy; LB, lysogeny broth; Eq., equation; 3D, three dimensional; CFU, colony forming unit; SD, standard deviation
⁎
Corresponding author.
E-mail address: mitalimadhumita5@gmail.com (M. Madhumita).

https://doi.org/10.1016/j.indcrop.2019.111578
Received 19 April 2019; Received in revised form 14 July 2019; Accepted 16 July 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
M. Madhumita, et al.
particular odour specific to betel leaf may be used for several medicinal
purposes such as to relieve pain and promote healing. It possesses
various important biological properties like antioxidant, antifungal,
antidiabetic, antiamoebic, anti-inflammatory, antimicrobial, and allied
properties (Guha, 2006 and Guha, 2007; Desai and Parikh, 2015).
Therefore, most of the chemical compounds derived from the plants
have a wide range of potential applications such as food, flavour, fra-
grance, and pharmaceutical industries etc (Thakker et al., 2018). As a
result, numbers of researches are going on the analysis of bioactive
compounds present in the EO.
Phyto-chemical compounds present in EO of plant materials are
important components of human diet that can regulate the oxidation
and stress related chronic diseases. To extract this EO from plant ma-
terials, several methods of extraction techniques such as hydro-dis-
tillation (HD), steam distillation, steam and water distillation, solvent
extraction, super critical extraction etc. were adopted (Dai et al., 2010;
Mason et al., 2011; Chemat et al., 2012). These techniques are widely
used to enhance the extraction efficiency and to identify the chemical
constitutes because of its simple and fast repeatability. However, HD
method was widely used to extract EOs from most of the plant matrix
that has gained much popularity in the present time with low cost and
environmental friendliness (Memarzadeh et al., 2015; Umar et al.,
2018). Up to now, there are several methods which have been widely
used for the identification of all phyto-chemical compounds present in
extracted EOs and these include gas chromatography- mass spectro-
metry (GC–MS), high-performance liquid chromatography (HPLC),
high-performance liquid chromatography with mass spectrometry
(LC–MS), nuclear magnetic resonance spectrometry (NMR), Fourier
transform infrared (FT-IR) spectroscopy etc. Gas chromatography- mass
spectrometry is a central useful analytical tool in the research field of
herbal medicines, especially for identification of various mixtures of
organic compounds present in isolated EOs (Matasyoh et al., 2007; Gu
et al., 2014). Now-a-days, more sophisticated vibration spectrometric
methods are followed such as FT-IR and FT-Raman etc. to find out the
chemotypes because of time and sample treatment (Daferera et al.,
2000). Fourier transform infrared spectrometry is a simple, rapid and
non-destructive method used for the determination of main components
and also identifies the functional group present in EO. It does not re-
quire any pre-treatment before the identification (Schulz et al., 2002;
Pappas et al., 2003).
Generally, contaminated foods caused by gram-positive and gram-
negative bacteria are harmful for the society as well as to industry. To
increase the resistance of microbial activity, spices and herbs play an
important safely role as food flavouring agent, as herbal medicine and
now known as “generally regarded as safe” (GRAS). Recently, EOs and
extracts of spices and herbs are considered in research study to inhibit
the growth of microbes (Valero and Salmeroj, 2003; Roby et al., 2013).
Microbial contamination is one of the key parameter to reduce the food
quality and shelf-life that causes several food borne diseases, deterio-
rates the food sensory properties and oxidation as well. Literature have
reported that food contaminated with various pathogenic bacteria such
as Pseudomonas aeruginosa, Staphylococcus aureus, and Mycobacterium
smegmatitis represents a serious health risk to human being (Gutierrez
et al., 2012; Diao et al., 2014). Therefore, there is a necessity to find
novel and safe natural antibacterial agents to extent shelf-life of foods
(Rai et al., 2011; Agarwal et al., 2012) and EO can be explored as an
antibacterial agent. Lots of reports are available on the microbial ac-
tivity of herbs extract and EO. However, no information is available for
the microbial study of cured betel leaf EO. Therefore, more research is
required on antimicrobial effects on fresh and cured leaf EO.
Several extraction variables such as extraction temperature, ex-
traction time, and material to solvent quantity were optimized to get
maximum extraction yield by using Box-Behnken design (BBD) coupled
with response surface methodology (RSM). Response surface metho-
dology, a valuable tool helps to determine the optimum values of ex-
perimental parameters and builds a mathematical model to evaluate the
2
Industrial Crops & Products 138 (2019) 111578
interaction effect between variables with a reduced number of experi-
ments (Maran et al., 2013; Muruganandam et al., 2017). To the best of
our knowledge, RSM application and optimization of combined use of
fresh and cured betel leaf EO yield treatment is not yet reported. The
composition of EO of betel leaf has been studied by a few researchers
but only from the fresh leaves. Till now, no study has been reported on
the extraction of EO from cured betel leaves. Therefore, it is not clear
whether there is any change in yield and composition of the oil or its
components due to the treatments imparted during curing of the betel
leaves, consequently selection of the best treatment for curing the betel
leaves remain partly in dark. However, according to literature survey,
identification of chemical components derived from cured betel leaves
have not been investigated yet. Therefore, firstly, the aim of the study
was to optimize the EO yield extracted from both fresh and cured betel
leaves using three factors BBD of RSM design. Secondly, the study was
aimed to determine the main constituents present in optimized fresh
and cured betel leaf EO that analyzed by the formation of spectral li-
braries by GC–MS. Thirdly, the aim of the present paper was to in-
vestigate the antimicrobial activity of EO and to study the morpholo-
gical mechanism of the optimized extract by scanning electron
microscopy (SEM) analysis.
2. Materials and methods
2.1. Materials and chemicals
Fresh and cured betel leaves of Bangla variety were collected from
Datan village, West Medinipore district of West Bengal, India. In order
to maintain the quality, both fresh and cured leaves were washed
thoroughly with distilled water to remove dirt. Both the leaves were
dried in a re-circulatory tray dryer for 2 h at 50 °C up to moisture
content (11.25%, d. b.). The dried leaves were crushed into powder
form and stored in a refrigerator about 4 °C to avoid any biochemical
changes before experimental analysis. Ethanol, methanol, lysogeny
broth (LB, L2897- powder for microbial growth medium), and LB agar
plate were purchased from Sigma Aldrich, Kolkata, India.
2.2. Extraction and isolation of EO
Two hundred grams of cleaned fresh and cured betel leaves was
hydro-distilled separately in triplicate with a Clevenger apparatus
(Guha, 2007). Leaves were placed into a 5 L size of round-bottom dis-
tillation flask. The round bottom flask was heated by heating mantle at
a temperature of 100ᵒC. During the extraction process, the steam and
vaporized oil were condensed into liquid form by a vertical condenser
and collected in the receiver tube. The volatile EO was then separated
from water by a separating funnel and collected in a measuring tube.
The collected EO was dried over anhydrous sodium sulphate until the
last traces of water had been removed, labelled in vials and stored in
refrigerator at 4ᵒC for further use. The EO yield of both leaves was
expressed as mean values on dry weight basis. The EO yield was de-
termined by the following Eq. (1):
Volume of extracted EO, (ml)
EO Yield = × 100; %
Dry weight of leaves, (g ) (1)
2.3. Experimental design and statistical analysis
Design expert software version 7.0 was applied to optimize the EO
yield extracted from fresh and cured betel leaves by HD method using
three independent extraction parameters. The independent parameters
i.e. extraction variables influence the EO yield extracted from plant
materials during extraction process. The effect of temperature and
solvent concentration on EO yield had been explored (Muruganandam
et al., 2017). Other independent parameters such as liquid to solid,
M. Madhumita, et al. Industrial Crops & Products 138 (2019) 111578

Table 1
Uncoded and coded independent variables used in RSM design for optimization
studies.
Independent variables Coded values
−1 0 +1
liquid to solid (mL/g), X1 30 40
Extraction time (h), X2 5 6
Particle size (mesh), X3 0 10
extraction time and particle size were selected for the present study to
explore the effect of these parameters on yield within the range of in-
vestigation. The selected three independent parameters were optimized
to minimise the variation during the extraction of process parameters
and to achieve a better suitable combination of factor levels that pro-
duce an optimum desirable response (extracted yield) which would
qualify the process for commercial application. Box-Behnken design
with RSM (Xu et al., 2013; Azmir et al., 2014) was employed to in-
vestigate the effect of extraction parameters such as liquid to solid (X1,
30:1-50:1 mL/g), extraction time (X2, 5–7 h), and particle size (X3, 0–20
mesh) on responses such as fresh leaf EO yield (FLEO, Y1) and cured leaf
EO yield (CLEO, Y2). The lower and higher levels of extraction variables
were selected on the basis of preliminary experiments and review of
literatures (Desai and Parikh, 2015; Muruganandam et al., 2017; Yuan
et al., 2019). Moreover, the powder mesh of P. betle was set as 0–20
mesh by doing preliminary trials on the basis of the methods described
by Memarzadeh et al., 2015 and Cui et al., 2018. This high range i.e.
from 0 to 20 mesh was selected to explore the effect of particles sizes of
betel leaf powder on the extraction of its EOs. All the ranges of selected
independent parameters were coded at three different levels (Table 1)
with three replications of the centre points. A BBD with seventeen
different experiments was generated with 5 replications at the centre
points (Table 2). A second order polynomial equation was used to de-
scribe the effects of different factors on response given as (Eq. 2):
k k−1 k k
Y = β0 + ∑ βi Xi + ∑ ∑ βij Xi Xj + ∑ βii Xi2 + ei
i=1 i=1 j=2 i=1
Analysis of variance was used to check the adequacy of best fit re-
sponse surface models and also to evaluate the significance of in-
dividual, quadratic and interaction terms in Eq. (2). p-value, F-value,
lack-of-fit test, and coefficient of determination (R2), predicted R2,
adjusted R2, and predicted residual error of sum square (PRESS) were
evaluated for sustainability, predictability, and adequacy of the devel-
oped second order regression equations. The insignificant terms
50
7 (p > 0.05) were removed and significant terms (p < 0.05) were re-
20 fitted to the equation to obtain a final reduced model. The three-di-
mensional (3D) response surface graphs of each interaction effect of
extraction variables were generated using Design Expert software and
to visualize the effect of each extraction parameter on EO yield.
2.3.1. Optimization and validation of models
The optimum level of HD extraction conditions was determined by
applying graphical and numerical optimization procedures to get the
maximum EO yield. The numerical and graphical optimization was
executed by the response optimizer and 3D response surface plots, re-
spectively to get an optimum level of the independent variables with
desired optimum response value. The response variable (EO yield, %)
was studied by using the optimized value at least three times to validate
the optimized data. Three dimensional response surfaces were plotted
to get a better visualization significant (p < 0.05) interaction effect of
HD variables (X1X2, X1X3, and X2X3) on the EO yield. The 3D plots were
generated by fixing one extraction variable constant at the centre points
and changing other two extraction variables within the experimental
range.
For validation of the developed model, both fresh leaf EO (FLEO)
and cured leaf EO (CLEO) was analyzed using optimum value of each
extraction variable. Both EO yield were developed and checked at least
three times using the optimized value to validate the optimised data. A
comparison was made between the experimental and predicted values
by using Tukey test (t-test) to check the variability (Mirhosseini et al.,
2009). The absolute error was calculated based on Eq. (3) as shown
below.
Experimental value − Predicted value
Absolute error = × 100; %
Predicted value (3)
(2)

Where, Xi values are independent variables affecting the response value
Y and β0, βi , βii, and βij are the regression coefficients for intercept,
linear, quadratic and interaction terms, respectively. The term, K re-
presents the number of variables. Analysis of variance (ANOVA) test
and regression equations were analyzed to determine the statistical
significance. All the experimental data were expressed as mean ±
standard deviation.
2.4. GC–MS analysis of EO
The chemical composition of EO extracted from both fresh and
cured leaves at different operating conditions was analyzed by GC–MS
that used to separate the compounds from volatile oil by gas chroma-
tograph (GC) component and then identifies those compounds at a
molecular level by the mass spectrometer (MS) component. For the

Table 2
Design matrix using BBD with experimental data for fresh and cured betel leaf EO yield.
Runs Independent Variables Response Variables

X1a, (mL/g) a
X2 , (h) a
X3 , (mesh) FLEO yieldb, (%) CLEO yieldb, (%)

1 30 (-1) 5(-1) 10(0) 0.1 ± 0.01 0.12 ± 0.02

2 40 (0) 6(0) 10(0) 0.18 ± 0.01 0.22 ± 0.02
3 30 (-1) 6(0) 0(-1) 0.11 ± 0.02 0.12 ± 0.01
4 30 (-1) 6(0) 20(+1) 0.11 ± 0.02 0.12 ± 0.01
5 40 (0) 7 (+1) 0(-1) 0.13 ± 0.01 0.15 ± 0.03
6 50 (+1) 7(+1) 10(0) 0.12 ± 0.01 0.13 ± 0.06
7 50 (+1) 6(0) 0(-1) 0.17 ± 0.01 0.2 ± 0.02
8 40 (0) 5(-1) 20(+1) 0.11 ± 0.03 0.12 ± 0.01
9 40 (0) 7(+1) 20(+1) 0.14 ± 0.11 0.16 ± 0.01
10 30 (-1) 7(+1) 10(0) 0.09 ± 0.06 0.1 ± 0.03
11 40 (0) 5(-1) 0(-1) 0.13 ± 0.01 0.14 ± 0.01
12 50 (+1) 5(-1) 10(0) 0.12 ± 0.01 0.13 ± 0.02
13 50(+1) 6(0) 20(+1) 0.12 ± 0.03 0.13 ± 0.01

a
X1 : Coded variable of liquid to solid; X2 : Extraction time; X3: Particle size.
b
Mean ± Standard deviation of experimental results.

3
M. Madhumita, et al.
characterization of volatile compounds, both FLEO and CLEO were
subjected to GC–MS (Thermo Scientific GC (TRACE™ 1300) and MS
(DSQ II) that equipped with a flame ionization detector. Twenty micro
litres of FLEO and CLEO were dissolved in 480 μL of methanol and 1 μL
of this solution was injected into GC–MS system to identify the chemical
compounds presented in EOs. The injection temperature and ion source
temperature of GC are set at 250 °C. The column temperature is held
isothermal at 70 °C for 2 min, and then programmed to 250 °C for
10 min. Helium was taken as carrier gas. An electron ionization mode
with 70 eV ionization energy was used for the GC–MS identification.
The sector mass analyzer was set to scan from 40 to 500 amu for 2 s
(Basak and Guha, 2015). Each individual quantified component of both
EO was identified by relative peak percent area, retention time and
mass fragmentation pattern using NIST mass spectral library (Filho
et al., 2017).
2.5. Anti-microbial activity evaluation
2.5.1. Microbial strains and sample preparation
Three different types of pathogenic microorganisms having strong
antibiotic resistance including Staphylococcus aureus (gram-positive
bacteria), Pseudomonas aeruginosa (gram- negative bacteria), and
Mycobacterium smegmatis (clinical drug resistant bacteria) were selected
for testing the antimicrobial activities of both FLEO and CLEO using
agar well diffusion method with slight modification (Ibrahim et al.,
2015; Rezaie et al., 2017). There is a necessity to evaluate the anti-
microbial effects of plant secondary metabolites against a range of
pathogens to discover safe and effective antimicrobials. Most of the EOs
extracted from leaves like Jatropha curcas L. (Babahmada et al., 2018),
Citrus aurantifolia L. (Al-Aamri et al., 2018), citronella (Timung et al.,
2016), lemongrass (Naik et al., 2010); citrus (Guo et al., 2018); Piper
betle L. (Aumeeruddy-Elalfi et al., 2015) etc. using HD method was
found effective against S. aureus, P. aeruginosa, and E. coli along with
other bacteria. Moreover, Guo et al. in 2018 reported that P. aeruginosa
and E. coli exhibited significant antimicrobial resistance against almost
all EO. Therefore, these bacterial species were selected for the anti-
microbial assay because of their resistive nature to inhibit the growth of
the bacterial strains. Phosphate Buffered Saline Tween-20 (1X PBST)
was prepared by dissolving 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4,
0.24 g of KH2PO4 and 2 mL of tween-20 in 800 mL of distilled water.
The solution was adjusted to pH of 7.2 and 1 L of volume was made by
adding distilled water and then sterilized by autoclaving.
2.5.2. Antimicrobial screening
The test pathogens (S. aureus, P. aeruginosa, and M. smegmatis) were
taken to test the antibacterial activity of both FLEO and CLEO following
the method of Ibrahim et al., 2015. A single colony was incubated in a
test tube containing 4 mL LB broth and incubated overnight at 37 °C.
From above solution, 1 mL culture was taken and centrifuged at
5000 rpm for 5 min. The pellet was re-suspended in 1 mL LB broth and
then again centrifuged at same rpm and time. Then OD of the culture
was taken at 600 nm and adjusted to 0.1 OD units. The formulation was
prepared by adding the above bacterial suspension to different con-
centrations (0.2 mL, 0.5 mL, and 1 mL) of EO samples and incubated at
37 °C for different time points (6 h and 24 h). The incubated samples
were serially diluted by 1X PBST with proper vertexing and plated on
LB plates by drops. After absorption of drops, the plates were taken for
incubation at 37 °C. To determine the antibacterial activity of FLEO
(sample 1) and CLEO (sample 2), the above mentioned concentrations
of oil were incubated with equal amount of chosen bacteria samples by
colony forming units (CFUs) assay in LB media in 96 well round bottom
plates. After respective time point’s bacteria were harvested at the in-
dicated time points and the number of CFUs were assayed by plating
diluted cultures on LB agar plates. Bacterial colonies were enumerated
after 6 and 24 h for S. aureus, P. aeruginosa, and M. smegmatis, bacteria
respectively. All the samples were plated in triplicate and values were
4
Industrial Crops & Products 138 (2019) 111578
averaged from three independent trials. The colony was counted and
the graph was plotted.
2.6. SEM analysis
The morphological changes in both fresh and cured leaves after HD
extraction were analyzed using scanning electron microscopy (SEM),
ZEISS EVO 6o (POLARON –SC7620). The tested extracted samples were
air dried and taken for the SEM analysis. The samples were gold sputter-
coated by cathode spraying before analysis to improve the quality of the
changes and then placed on a specimen holder to get the micro struc-
ture. The analysis was done for examining the microstructure of leaf
surfaces at high resolution and to study the effect of extraction method
on leaves matrix.
2.7. Statistical analysis
All the experiments were performed in triplicate on three separate
samples. Mean values and standard deviations (mean ± SD) were ob-
tained from those triplicate trials.
3. Results and discussion
3.1. Regression analysis and model fitting
Response surface methodology coupled with BBD, a modelling de-
sign was employed to estimate the higher impact of extraction para-
meters (X1, X2, and X3) on the responses Y1 and Y2 (maximum FLEO and
CLEO Yield) that extracted from fresh and cured betel leaves. Analysis
of variance and regression analysis were employed to test the effect of
parameter terms (linear, interaction, and quadratic term) and also to
get the better accuracy, credibility, and goodness of fit of the quadratic
model Eq. (2) obtained from the experimental results. The p- value <
0.05 and F-value indicates the statistical significance of the ANOVA
model and examined the estimated regression models and corre-
sponding regression coefficients (Table 3 and 4). From the ANOVA
Tables, it was concluded that both FLEO and CLEO model were sig-
nificant having p-value 0.0019 and 0.018, respectively. According to
some literature studies, the value of determination coefficeient
(R2) > 0.75 showed the adequacy of the model (Subramonian et al.,
2015). In the present study, R2 for FLEO and CLEO were found to be
0.9376 and 0.8751, respectively that indicates that both the models
were adequate in this optimization study. Comparing both models, it
was ensured that the FLEO regression model was more satisfactorily
fitted to the experimental data than CLEO having high p-value. The
adjusted R2 of both the models were very close to their respective R2
values (0.8574 and 0.7144) which indicates a high degree of correlation
between observed and predicted values (Chen et al., 2006). The Rpred2
of FLEO and CLEO (0.4256 and 0.0289) was in reasonable agreement
with Radj2 of FLEO and CLEO (0.8574 and 0.7144) that indicates a high
degree of correlation. Further, the adequate precision for both the
models were 9.17 and 6.17, respectively that indicates that the model
was adequate. The lack of fit for both FLEO and CLEO models
(p = 0.3429 and 0.4323) for extraction yield were non- significant
(p > 0.05) that indicates that the mathematical model was satisfactory
for prediction of both yield (Prasad et al., 2011). Both FLEO and CLEO
models were accurate and satisfactory due to the significant value of R2
and non-significant lack-of fit (p > 0.05). All these statistical para-
meters of the ANOVA tables show the better accuracy, fitness and re-
liability of both the models.
Two second order polynomial mathematical models for FLEO and
CLEO were obtained by conducting the multiple regression analysis.
These mathematical models were applied to develop a relationship
between independent and response variables. Tables 3 and 4 showed
that, the quadratic terms (X12 and X22) found to be significant for both
FLEO and CLEO. The independent variable, X1 of FLEO had significant
M. Madhumita, et al. Industrial Crops & Products 138 (2019) 111578

Table 3
Analysis of variance (ANOVA) for the fitted quadratic polynomial model of fresh betel leaf EO Yield.
Source SSa dfa MSa F-value p-value

−3
Model 0.013 9 1.419 × 10 11.69 < 0.0019 Significant
X1 1.8 × 10−3 1 1.8 × 10−3 14.82 < 0.0063 Significant
X2 5 × 10−5 1 5 × 10−5 0.41 0.5415
X3 4.5 × 10−4 1 4.5 × 10−4 3.71 0.0956
X1 X2 2.5 × 10−5 1 2.5 × 10−5 0.21 0.6637
X1 X3 6.25 × 10−4 1 6.25 × 10−4 5.15 0.0576
X2 X3 2.25 × 10−4 1 2.25 × 10−4 1.85 0.2156
X12 4.112 × 10−3 1 4.112 × 10−3 33.86 0.0007 Significant
X22 4.112 × 10−3 1 4.112 × 10−3 33.86 0.0007 Significant
X32 5.329 × 10−4 1 5.329 × 10−4 4.39 0.0744
Lack of Fit 4.5 × 10−4 3 1.5 × 10−4 1.5 0.3429 Not Significant
R2 0.9376
Radj2 0.8574
RPred2 0.4256
Adeq precision 9.17

a
SS = Sum of Square, df = Degree of freedom, MS = Mean square, and X1 = Liquid to solid (mL/g), X2= Extraction time (h), X3= Particle size (mesh).

Table 4
Analysis of variance (ANOVA) for the fitted quadratic polynomial model of Cured betel leaf EO Yield.
Source SSa dfa MSa F-value p-value

−3
Model 0.021 9 2.315 × 10 5.45 0.0180 Significant
X1 2.112 × 10−3 1 2.112 × 10−3 4.97 0.0610
X2 1.125 × 10−4 1 1.125 × 10−4 0.26 0.6227
X3 8 × 10−4 1 8 × 10−4 1.88 0.2124
X1 X2 1 × 10−4 1 1 × 10−4 0.24 0.6424
X1 X3 1.225 × 10−3 1 1.225 × 10−3 2.88 0.1334
X2 X3 2.25 × 10−4 1 2.25 × 10−4 0.53 0.4905
X12 6.737 × 10−3 1 6.737 × 10−3 15.85 0.0053 Significant
X22 6.737 × 10−3 1 6.737 × 10−3 15.85 0.0053 Significant
X32 1.289 × 10−4 1 1.289 × 10−4 3.03 0.1251
Lack of Fit 1.375 × 10−3 3 4.583 × 10−4 1.15 0.4323 Not Significant
R2 0.8751
Radj2 0.7144
RPred2 0.0289
Adeq precision 6.166

a
SS = Sum of Square, df = Degree of freedom, MS = Mean square, and X1 = Liquid to solid (mL/g), X2= Extraction time (h), X3= Particle size (mesh).

Fig. 1. Response surface plots showing interaction effect of different parameters on (a1-a3) fresh leaf essential oil (FLEO) yield and (b1-b3) Cured leaf essential oil
(CLEO) Yield.

5
M. Madhumita, et al. Industrial Crops & Products 138 (2019) 111578

Fig. 2. Predicted yield vs actual yield (a) fresh leaf essential oil (FLEO) yield (b) cured leaf essential oil (CLEO) yield.

Table 5 3.2. Effect of independent variables on EO yield

Comparison between experimental and predicted values based on the final re-
duced model. The three dimensional (3D) responses for fresh and cured betel leaf
Std Run FLEO Yield (%) CLEOYield (%) EO yield are shown in Fig. 1 which indicated the effects the significant
terms on the tested response. The extraction parameters such as liquid
Y0 Yi Y0- Yi Y0 Yi Y0- Yi to solid (X1, 30:1-50:1 mL/g), extraction time (X2, 5-7 h) and particle
size (X3, 0–20 mesh) influenced the fresh and cured betel leaf EO yield.
1 0.1 0.11 −0.01 0.12 0.12 0
2 0.18 0.16 0.02 0.22 0.18 0.04 The 3D curves showed the main and interaction effect between two
3 0.11 0.10 0.01 0.12 0.12 0 extraction parameters on the EO yield by maintaining the third para-
4 0.11 0.11 0 0.12 0.12 0 meter at zero level. Fig. 1 (a1-a3) and (b1-b3) exhibited the main (X1,
5 0.13 0.17 −0.03 0.15 0.2 −0.05 X2, and X3) and interaction effect (X1X2, X1X3 and X2X3) on the yield of
6 0.12 0.15 −0.03 0.13 0.18 −0.05
FLEO and CLEO, respectively. At constant particle size of 10 mesh, li-
7 0.17 0.16 0.01 0.2 0.18 0.02
8 0.11 0.15 −0.04 0.12 0.17 −0.05 quid to solid parameter affected the FLEO and CLEO yield in a linear
9 0.14 0.15 −0.01 0.16 0.17 −0.01 and quadratic manner both while extraction time significantly influ-
10 0.09 0.10 −0.01 0.1 0.12 −0.02 enced in a quadratic manner only (Fig. 1 (a1 and b1)). The extraction of
11 0.13 0.11 0.02 0.14 0.12 0.02
FLEO and CLEO achieved the highest value such as 0.18% and 0.22%
12 0.12 0.11 0.01 0.13 0.12 0.01
13 0.12 0.15 −0.03 0.13 0.17 −0.04
when liquid to solid and extraction time varied from 35:1 to 45:1 mL/g
and 5 to 6.5 h, respectively and thereafter, the FLEO and CLEO yield
Y0 : Experimental value; Yi : Predicted value; Y0- Yi: Residue value. was gradually decreased. However, the interaction effect (X1X2) was
found to be not significant. Fig. 1 (a2 and b2) showed that the 3D re-
effect among all the linear model terms. However, the linear terms (X2 sponse curve unfolded the main and interaction effect (X1X3) of liquid
and X3), interaction model terms (X1X2, X1X3, and X2X3), quadratic to solid ratio and particle size on FLEO and CLEO yield at constant
term (X32) were found to be non-significant for both the models. Ac- extraction time (6 h). The extraction yield of FLEO and CLEO sig-
cording to Tables 3 and 4, the linear term (X1) and the quadratic terms nificantly increased with increase in liquid to solid from 32:1 mL/g to
(X12 and X22) showed significant effects (p < 0.05). The final quadratic 45:1 mL/g, and particle size from 5 mesh to 15 mesh and thereafter
polynomial equations for FLEO and CLEO were formulated by using the both the EO yield was decreased. On the other hand, the increase of
regression coefficient values with ignoring insignificant terms and are particle size accelerated the mass transfer, increasing the extraction
given by the following Eqs. 4 and 5: yield. The reason behind this, the smaller particle size enlarges surface
area which in turn increased the EO yield whereas too smaller size
Y1 (FLEO) = +0.17 + 0.015 X1 + 2.5×10−3X2 -7.5×10−3X3
particles can decrease the EO yield due to the inhomogeneous extrac-
+2.5×10−3X1X2 – 0.013 X1X3 + 7.5×10−3X2X3 -0.031X12 -0.031X22
tion (Zheljazkov et al., 2014; Kusuma and Mahfud, 2015). Moreover,
-0.011X32 (4)
extraction time showed a quadratic effect whereas particle size did not
Y2 (CLEO) = +0.2 + 0.016 X1 + 3.75×10−3X2 -0.01X3 affect significantly the yield of FLEO and CLEO at constant liquid to
+5×10−3X1X2 – 0.018 X1X3 + 7.5×10−3X2X3 -0.04X12 -0.04X22 - solid ratio of 40:1 mL/g (Fig. 1 (a3 and b3)). Results showed that the
0.017X32 (5) increment in extraction time from 5 h to 6.5 h with particle size from 5
mesh to 15 mesh, enhanced the extraction yield of both FLEO and CLEO
From the above quadratic regression model equations (Eqs. 4 and yield. Initially, EO yield increased significantly as extraction time in-
5), it was observed that the linear variables (X1 and X2) and the inter- creases because of the more extraction time, oil pockets are diffused out
action variables (X1X2 and X2X3) have a positive effect on the extraction due to the higher boiling point (Zhang et al., 2012). The extraction
yield, whereas other terms has a negative effect. yields started declining when the extraction time and particle size
surpassed 7 h and 15 mesh. Due to the inadequate cooling time of the

6
M. Madhumita, et al.
Fig. 3. GC–MS profile of optimized betel leaf essential oil (EO). (A) Fresh Betel Leaf EO (B) Cured Betel Leaf EO.
apparatus, minimum oil yield was obtained at higher duration time
(Belhachat et al., 2018). In addition, the regression model was used to
explore the interaction effects of three independent parameters for
extraction of FLEO and CLEO yield. According to the 3D response
surface analysis, the linear term (X1) and quadratic terms (X1 and X2)
had significant effect on the extraction yield. So, both FLEO and CLEO
models were found to be significant (p = 0.0019 and p = 0.018), re-
spectively.
The effect of extraction variables and its interaction on the yield of
7
Industrial Crops & Products 138 (2019) 111578
fresh and cured betel leaves essential oil was mathematically modelled
in terms of second order polynomial equation. The second order poly-
nomial model adequacy was investigated by comparing and plotting the
experimental value and predicted values of extracted FLEO and CLEO
yield (Fig. 2). The fitted line plot represents the deviation of the results
of experimental data predicted values. Also this plotted figure helps to
investigate individual data point of each variable (Zhang et al., 2012).
Fig. 2 revealed that the experimental and predicted values derived from
the model were so close to the straight fitted line and there was no
M. Madhumita, et al. Industrial Crops & Products 138 (2019) 111578

Table 6
Volatile chemical compounds of CLEO extracted by HD at different operating conditions.

a
RI Compounds 1 2 3 4 5 6 7 Central Points 8 9 10 11 12

4.82 Cis- geraniol 0.256 0.263 0.263 0.262 0.263 0.262 0.262 0.261 ± 0.01 0.261 0.26 0.26 0.263 0.263
4.93 Cis-ocimene 0.436 0.436 0.435 0.434 0.436 0.435 0.434 0.436 ± 0.02 0.436 0.435 0.434 0.434 0.434
5.36 Caryophyllene oxide 0.62 0.62 0.62 0.6 0.58 0.58 0.65 0.62 ± 0.02 0.62 0.62 0.6 0.63 0.63
5.43 Pyrimidine 0.642 0.642 0.64 0.64 0.642 0.642 0.642 0.64 ± 0.01 0.64 0.641 0.641 0.64 0.64
5.95 2-3-isopropyl-4-methyl-pent-3 0.82 0.82 0.81 0.81 0.81 – 0.83 0.82 ± 0.02 0.82 0.81 0.81 0.82 0.82
cyclobutanone
6.14 Cumene 0.84 0.87 0.87 0.871 0.871 0.872 0.873 0.873 ± 0.01 0.873 0.875 0.875 0.873 0.873
6.2 γ- murolene 0.72 0.737 0.738 0.738 0.737 0.737 0.737 0.736 ± 0.01 0.736 0.738 0.738 0.737 0.737
7.1 Citral 0.657 0.661 0.663 0.663 0.663 0.662 0.662 0.661 ± 0.01 0.661 0.662 0.662 0.661 0.662
7.35 Chavicol 1.52 1.52 1.52 1.523 1.524 1.524 1.524 1.565 ± 0.02 1.523 1.523 1.523 1.52 1.52
7.42 Anethole 2.545 2.645 2.646 2.647 2.649 2.649 2.648 2.648 ± 0.01 2.649 2.646 2.647 2.647 2.646
7.96 Estragole 17.124 15.124 15.123 15.123 15.123 15.122 15.122 17.122 ± 0.02 15.123 15.121 15.085 15.123 15.122
8.05 Eugenol 44.137 44.142 44.142 44.113 44.143 44.143 44.14 44.14 ± 0.03 44.14 44.141 44.101 44.142 44.142
8.68 Linalool 13.247 13.246 13.246 13.182 13.244 13.244 13.243 13.243 ± 0.01 13.244 13.245 13.194 13.244 13.245
8.87 α- copaene 5.338 5.341 5.342 5.342 5.343 5.343 5.34 5.341 ± 0.01 5.341 5.341 5.343 5.344 5.342
9.52 Chavibetol 0.563 0.564 0.564 0.562 0.562 0.562 0.564 0.58 ± 0.01 0.563 0.563 0.564 0.58 0.56
9.91 3-methoxy cinnamaldehyde 0.645 0.654 0.654 0.655 0.655 0.656 0.655 0.655 ± 0.01 0.654 0.656 0.655 0.654 0.655
9.99 Caryophyllene 1.125 1.127 1.124 1.126 1.126 1.126 1.125 1.125 ± 0.02 1.124 1.125 1.124 1.126 1.124
10.82 Viridiflorene 0.788 0.791 – 0.79 0.792 0.79 0.792 0.792 ± 0.01 0.791 0.791 0.79 0.79 0.792
11.28 Farnesene epoxide 1.05 1.052 – 1.053 1.053 1.053 1.054 1.052 ± 0.01 0.053 1.053 1.052 1.052 1.053
11.77 3-cyclohexene-1-methanol 1.2 1.23 1.23 – 1.210 1.21 1.22 1.23 ± 0.01 0.24 1.22 1.22 1.21 1.21
12.0 Aciphyllene 0.55 0.55 0.55 0.551 – 0.552 0.551 0.552 ± 0.02 0.551 0.552 0.55 0.551 0.552
12.33 4- butylbenzyl alcohol 0.4 0.42 0.42 0.41 0.400 0.4 0.41 0.42 ± 0.01 0.43 0.41 0.41 0.4 0.4
13.24 β- cubebene 0.5 0.52 0.52 0.521 0.521 0.521 0.525 0.525 ± 0.01 0.524 0.524 0.523 0.524 0.523
14.11 Acetyleugenol 0.222 – 0.221 0.221 0.222 0.222 0.221 0.221 ± 0.02 0.22 0.22 0.222 0.221 0.21
15.35 Elemene 0.152 0.152 0.154 0.154 0.155 0.154 0.153 0.152 ± 0.01 0.152 0.153 0.154 – 0.153
16.46 P-anisaldehyde 0.148 0.151 0.15 0.15 0.151 0.152 0.155 0.153 ± 0.02 0.153 0.152 0.15 0.15 0.151
17.09 methyl acetate 0.235 0.237 0.237 0.237 0.238 0.238 – 0.237 ± 0.03 0.237 0.238 0.238 0.237 0.237
18.55 Allo-aramaadendrene epoxide 0.136 0.155 0.156 0.156 0.156 0.156 0.154 0.154 ± 0.01 0.155 0.155 0.156 0.154 0.155
19.95 cis- verbenol 0.06 0.06 0.06 0.058 0.060 0.06 0.058 0.057 ± 0.02 0.057 0.058 0.058 0.059 0.058
21.25 Eucalyptol 0.058 0.068 0.07 0.07 0.070 0.07 0.065 0.065 ± 0.01 0.067 0.067 0.068 0.069 0.068
23.03 α- limonene 0.02 0.18 0.18 0.02 0.020 0.19 – 0.18 ± 0.01 0.18 0.18 0.19 0.02 0.02
24.09 Ledene 0.054 0.064 0.064 0.063 0.064 0.063 0.063 0.064 ± 0.01 0.064 0.063 0.063 0.064 0.064
25.44 γ- terpinene 0.023 0.025 0.025 0.025 0.025 0.024 0.024 0.023 ± 0.03 0.023 0.024 0.024 0.023 0.024
Total identified 96.831 95.067 93.437 93.77 94.508 94.414 94.896 97.343 93.305 95.262 95.124 94.962 95.085

a
RI: Retention index.

significant differences were found. This predicted vs. actual plotted
results also concluded that the developed response surface model was
suitable to optimize the conditions for the extraction of both fresh and
cured betel leaf EO.
3.3. Optimization and validation of the models
conditions were experimentally validated to confirm the adequacy of
both FLEO and CLEO models. Table 5 revealed the comparison analysis
of the experimental results with the predicted values of the response
variables and it was found that the absolute residual error for the de-
pendent variables of FLEO and CLEO were ranged from 0 to 0.02% and
0 to 0.04%, respectively. Hence, both the models were adequate for the
optimization process conditions that obtained by BBD.

Both FLEO and CLEO yield at each experimental runs were opti-
mized and explored by RSM. From Table 2, it was observed that for
FLEO and CLEO, the maximum and minimum yields were found to be in
run number 2 and 10, respectively. In conclusion, based on the ANOVA
and 3D response surface analysis, the optimum extraction parameters
for both FLEO and CLEO were found to be same such as 40:1 mL/g of
liquid to solid, 6 h of extraction time and 10 mesh of particle size. At
this optimized conditions, a maximum yield of 0.18 ± 0.01% (FLEO)
and 0.22 ± 0.02% (CLEO) were obtained. The higher yield was ob-
tained in CLEO because of the curing treatment that changed the leaf
green to yellow makes the leaf tissue loose and soft for enhanced dif-
fusion (Lucchesi et al., 2004). Thus, the EO diffused out easily from the
leaves at higher boiling point which in turn increased the yield sig-
nificantly. The similar findings have also been reported by Guha, 2007
that extracted EO from Mitha, Bangla and Sanchi varieties of betel
leaves were about 2.0%, 1.7% and 0.8%, respectively. Further, the
reasons behind these changes also depend on the variations in the cli-
mates, ecological environments, growth conditions and agronomic
management practices in the different cultivation sites (Ibrahim et al.,
2015).
The optimum response and condition values were obtained by the
model Eq.s 4 and 5 and confirmed through a two-sample t-test to verify
the correctness of the equations. The above mentioned optimal
3.4. Bioactive compounds by GC–MS
Essential oil isolated from fresh and cured betel leaves by HD
method using Clevenger-type apparatus gave yellowish brown colour
with a yield of 0.18 ± 0.01% and light whitish colour with a yield of
0.22 ± 0.02% (v/w), respectively based on dried weight. The ex-
tracted FLEO and CLEO yielded at different operating experimental
conditions were subjected to GC–MS analysis to identify and char-
acterize the chemical composition. Fig. 3A and 3B revealed a total of 33
and 30 bioactive compounds that representing 98.41% and 97.34% of
the optimized FLEO and CLEO that could have contributed to the var-
ious aromatic and medicinal properties of the plant. It can be seen that
identified compounds of FLEO and CLEO were almost similar in their
composition but in different percentage. Five major compounds in-
cluding eugenol (46.02% and 44.14%), estragole (15.12% and
17.12%), linalool (11.62% and 13.25%), α-copaene (6.54% and
5.34%), and anethole (2.62% and 2.65%) were found in FLEO and
CLEO, respectively. Moreover, chavicol (1.4% and1.52%), car-
yophyllene (1.12% and1.125%), farnesene epoxide (1.12% and 1.05%),
and 3 cyclohexane 1-methanol (1.2%) were also identified. In addition,
both FLEO and CLEO also contained considerable amounts of various
minor constituents of compounds whose contribution was less than

8
M. Madhumita, et al.
Fig. 4. Dose dependent killing of bacteria by optimized EO of both fresh and cured betel leaves. (A) Mycobacterium smegmatis (6 incubation hours) (B) Mycobacterium
smegmatis (24 incubation hours), (C) Pseudomonas aeruginosa (2 incubation hours) (D) Pseudomonas aeruginosa (6 incubation hours), (E) Staphylococcus aureus (6
incubation hours) (F) Staphylococcus aureus (24 incubation hours). Sample 1 (FLEO) and sample 2 (CLEO) incubated with bacterial strains, respectively.
10%. The main chemical constituents of FLEO and CLEO are mainly
oxygenated monoterpenes, sesquiterpenes, hydrocarbons, and also their
derivatives having low molecular weight. The sesquiterpene hydro-
carbons such as α-pinene, α-copaene, caryophyllene, and monoterpene
hydrocarbons such as cubebene, α-limolene, murolene, elemene etc.
with less percentage were found in both EOs. It has been reported that
chavibetol, estragole, β-cubebene, chavicol, and caryophyllene are the
major components of EO of Tamluk mitha variety betel leaves. The
difference in chemical composition of both FLEO and CLEO depends on
the several factors such as plant variety, growth stage, harvesting time,
climatic conditions, country of region, ecological, and geographical
locations (Abdelli et al., 2016).
Cured betel leaf EO found to be rich in estragole, linalool, and an-
ethole that can be mostly used in food and pharmaceutical industries.
The reason behind these changes may be due to the effect of smoke and
heat treatment imparted during the curing process. These compounds
have several valuable medicinal purposes. Linalool and eugenol as
oxygenated monoterpenes impart a strong characteristic aroma that
used in fragrances, cosmetics, shampoos, detergents etc. Furthermore,
these compounds possess antibacterial, antifungal, and insecticidal
properties that helps as a natural disinfecting solution (Abdelli et al.,
2016). This analysis was adapted to explore the impact of curing
treatment on the volatile components. Therefore, chemical compounds
of CLEO extracted at different operating conditions were evaluated and
presented in Table 6. It can be seen that the composition of CLEO was
9
Industrial Crops & Products 138 (2019) 111578
almost constant over the range of experimental conditions studied.
Table 6 revealed that Run no. 8 has a total 33 of compounds that
constitute 98.34% of CLEO whereas Run no. 3 explored 31 compounds
that made up 96.44% of total CLEO. Similar results were found in ve-
tiver EO extracted by supercritical fluid extraction method (Danh et al.,
2009; Chu and Kemper, 2011).
3.5. Antibacterial activity of EO
The antibacterial activity of extracted FLEO (sample 1) and CLEO
(sample 2) was assessed by the killing assay against three selected
bacteria such as gram-positive (S. aureus), gram-negative (P. aerugi-
nosa), multidrug resistant bacteria (M. smegmatis) with different time
interval by colony forming unit (CFU) assay (Fig. 4). This CFU analysis
of microbial growth for both FLEO and CLEO were noted in Fig. 4 that
depends on the concentration of EO. It was observed from Fig. 4 that EO
of both fresh and cured betel leaves have a significant bactericidal effect
on all strains used in this study. Among all the tested samples, M.
smegmatis bacteria were found to be more susceptible to 0.5 and 1 mL of
CLEO after 24 h incubation period. CLEO caused 12 CFU×104 reduc-
tions (Fig. 4A) within 6 h and 1 CFU×104 reduction within 24 h
(Fig. 4B). Therefore, CLEO exposure to 24 h exhibited significant anti-
bacterial activity against all the selected bacteria. Treatment with doses
of 1 mL of FLEO caused 10 CFU×104 reductions after 6 h incubation
period and 1 CFU×104 reductions after 24 h exposure period. It was
M. Madhumita, et al.
Fig. 5. SEM analysis of fresh and cured betel leaves after HD treatment. (A-a)
view of extracted fresh leaves at 500 and 1000 magnification (B-b) view of
extracted cured leaves at 500 and 1000 magnification.
10
Industrial Crops & Products 138 (2019) 111578
resulted that 1 mL of CLEO exposure to 24 h incubation period had a
strong anti-bactericidal effect than 6 h treatment period. M. smegmatitis
bacteria are more susceptible to antimicrobials having one pepti-
doglycan outer thick layer that consisting of two layers of polymers of
sugar and amino acid with one layer of proteins (Arias et al., 2004;
Prakash et al., 2010). It was also observed that there is no significant
difference in case of FLEO and CLEO against P. aeruginosa population.
From Fig. 4C and D, it was noticed that exposure to 1 ml of CLEO were
found to be 95 CFU×104 and 54 CFU×104 P. aeruginosa population
reduction after 2 h and 6 h incubation period, respectively. After 6 h of
incubation period, approximately 23 CFU×104 and 21 CFU×104 of S.
aureus reduction was found in 1 mL of FLEO and CLEO, respectively
(Fig. 4E). Similarly, after 24 h, approximately 21 CFU×104 and 38
CFU×104 of S. aureus reduction was found in 1 mL of FLEO and CLEO,
respectively (Fig. 4F). There were no viable colonies were found in
CLEO at the dose of 1 mL after 6 h and 24 h treatment period.
According to literature survey, there is a correlation between the
antibacterial activity of EOs and their chemical composition. In EO,
most of the antimicrobial activity is attributed to oxygenated terpenoids
like alcohols and phenolic terpenes (Chu and Kemper, 2011). Some of
the researchers reported that EO exhibited the highest antibacterial
activity due to the presence of some compounds such as cinnamalde-
hyde, citral, carvacrol, eugenol etc (Adrar et al., 2016; Yuan et al.,
2019). Lavender EO exhibited strong antibacterial activity against
S.aureus, E. Coli, and C. Albicans with MIC values of 1200 μg/mL,
2000 μg/mL and 1000 μg/mL, respectively (Yuan et al., 2019). The MIC
of betel leaf EO (Piper betle L. var. Tamluk mitha) against P. expansum
was found to be 0.74 μl/mL (Basak and Guha, 2015). From the result, it
was noticed that all the strains of S. aureus and P. aeruginosa bacteria
were found to be very sensitive to both FLEO and CLEO and also these
had little influence on growth inhibition. Prakash et al. (2010) revealed
that eugenol (63.39%) and acetyleugenol (14.05%) were the main
components in EO of betel leaf (cv. Magahi) which are responsible for
antifungal activity. Since the biological activity of EOs is often attrib-
uted to its major components like eugenol, linalool etc that were
evaluated in this study under experimental conditions (Table 6). Lina-
lool, a monoterpenoid compound, exhibited strong antibacterial ac-
tivity against the tested strains like E. coli, S. Aureus and P. Aeruginosa
(Marchese et al., 2017). In the present study, linalool was found to be
highest in CLEO that acts a strong antibacterial activity. Diao et al.
(2014) reported that monoterpene or sesquiterpene hydrocarbons and
their oxygenated derivatives of extracted EO exhibit potential anti-
microbial activities. Moreover, the EO extracted from H. Coronarium
rhizome found to be rich in linalool exhibited antibacterial activity
against five pathogenic bacteria (Prakash et al., 2010). CLEO exhibited
strong antibacterial activity that might be due to the presence of lina-
lool compound in higher percentage than other compounds (13.25%).
There was a significant reduction in microbial counts at 6 h and 24 h
exposure and after 24 h exposure there was a complete inhibition of cell
viable counts. This may be due to the presence of eugenol compound in
higher percentage showing high antibacterial activity by disrupting the
bacterial membrane activity (Onawunmi et al., 1984; Nouri et al., 2014;
Desai and Parikh, 2015).
3.6. Morphological study by SEM
Morphological changes in the leaf matrix after extraction was ana-
lyzed by scanning electron microscopy analysis (SEM) as shown in
Fig. 5. Fig. 5 (A, a, B, b) described the structure of both extracted fresh
and cured betel leaves that were verified by (SEM) at 500 and 1000
magnification. The result of SEM showed a comparison between the
effects of extraction conditions on all samples. Small changes were
observed in Fig. 5B and 5b because of the curing treatment of leaves. It
was observed that there was a slight rupture and disruption of the
structure of vegetal cell on the surfaces of cured sample compared with
fresh that enhanced the extraction yield. The SEM images revealed that
M. Madhumita, et al.
fibres of fresh extracted leaves are less damaged and intense than cured
leaves because due to smoke treatment of cured leaves the tissues are
more disrupted and corrugated. Fig. 5A and 5a showed the presence of
fibrous networks that gives rough surface and porosity of the cells
which are clearly visible. The reason of no rupture cell wall in extracted
fresh leaves depends on the hydro-diffusion phenomenon that does not
allow the volatile oils to escape (Ferhat et al., 2006; Blazekovic et al.,
2018). After the extraction, the leaves material got wrinkled and
porous. The microscopy images of cured leaves showed highly porous
structure indicating loose and rough than fresh leaves because during
curing treatment the fibres of betel leaves get more collapsed and se-
parated due to this chlorophyll degradation (Fig. 5B and 5b). The main
reason behind this chlorophyll degradation is smoke treatment. From
theses morphological changes, it was observed that there is a significant
change in the structure of the plant material due to the extraction
method which contributes to the increase in its surface, improving the
mass transfer rate. In the current studies, SEM images confirmed that
extracted fresh leaves showed a non-porous, fibrous, smooth surface in
nature that can improve the extraction of bioactive compounds while
cured leaves showed a non-porous and rough surface in nature (Riela
et al., 2008; Desai and Parikh, 2015).
4. Conclusions
Response surface methodology with BBD was successfully employed
to investigate the effect of optimum extraction conditions (liquid to
solid, extraction time and particle size) on response yields (fresh and
cured betel leaf EO yield). The optimum conditions of maximum FLEO
yield (0.18 ± 0.01%) and CLEO (0.22 ± 0.02%) was found to be
40:1 mL/g of liquid to solid, 6 h of extraction time and 10 mesh of
particle size. Gas chromatography mass spectrometry analysis revealed
that the extracted essential oil from cured betel leaves possesses better
biochemical activity than the fresh betel leaves. Estragole, linalool,
chavicol, and caryophyllene compounds were identified with higher
percentage of biomarker in essential oil of cured betel leaves than the
fresh leaves. The anti-microbial activity results of both FLEO and CLEO
revealed that M. smegmatis bacteria were found to be more susceptible
to CLEO among all the selected bacteria, S. aureus, P. aeruginosa, and M.
smegtitis. The cured betel leaf EO was proved to have strong anti-
microbial activity that allows it to be used by the pharmaceutical and
food industries as natural preservatives. Morphological study was
useful to understand the extraction mechanism. It was concluded that
betel leaf essential oil having valuable bioactive compounds can be
used for the quality evaluation for herbal product which will become a
key technique for further future use.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors would like to thank Indian Institute of Technology
Kharagpur, West Bengal, India for financial support in the form of re-
search fellowship and also for the required lab and library facilities. The
author would also like to thank School of Biotechnology, KIIT,
Bhubaneswar, Odisha providing lab facilities for the research work.
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