Separation Science and Technology

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsst20

Green extraction of ethnomedicinal compounds

from Cymbopogon citratus Stapf using hydrogen-
bonded supramolecular network

Zubera Naseem , Muhammad Zahid , Muhammad Asif Hanif & Muhammad

Shahid

To cite this article: Zubera Naseem , Muhammad Zahid , Muhammad Asif Hanif & Muhammad
Shahid (2020): Green extraction of ethnomedicinal compounds from Cymbopogon�citratus Stapf
using hydrogen-bonded supramolecular network, Separation Science and Technology, DOI:
10.1080/01496395.2020.1781894

To link to this article: https://doi.org/10.1080/01496395.2020.1781894

Published online: 24 Jun 2020.

Submit your article to this journal

Article views: 14

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=lsst20
SEPARATION SCIENCE AND TECHNOLOGY
https://doi.org/10.1080/01496395.2020.1781894

Green extraction of ethnomedicinal compounds from Cymbopogon citratus Stapf

using hydrogen-bonded supramolecular network
Zubera Naseema, Muhammad Zahida, Muhammad Asif Hanifa, and Muhammad Shahidb
a
Department of Chemistry, University of Agriculture, Faisalabad, Pakistan; bDepartment of Biochemistry, University of Agriculture, Faisalabad,
Pakistan

ABSTRACT ARTICLE HISTORY

Deep eutectic solvent (DES) was synthesized using choline chloride/glycerol for the extraction of Received 23 August 2019
ethnomedicinal compounds from Cymbopogon citratus by maceration and ultrasound-assisted Accepted 8 June 2020
extraction (UAE). The optimization of extraction parameters and their interactive influence were KEYWORDS
evaluated by response surface methodology. Extracts were characterized using different antiox- Deep eutectic solvent;
idant and antimicrobial activities. Different phenolics/flavonoids were identified by LC-ESI-MS. The Cymbopogon citrates;
significantly enhanced extraction yield was obtained using DES in comparison with aq. methanol. response surface
Higher values of TPC, TFC, and DPPH inhibition were obtained (135 mg GAE/g, 91 mg QE/g, and methodology; ultrasound-
93%, respectively) for DES by applying UAE as compared to maceration. assisted extraction; LC-ESI-
MS
Abbreviations: DES: Deep eutectic solvent; ChCl:Gly: Choline chloride: glycerol; UAE: Ultrasound-
assisted extraction; TPC: Total phenolic content; TFC: Total flavonoids content; DPPH: 2, 2-diphenyl-1-
picrylhydrazyl; RSM: Response surface methodology; ANOVA: Analysis of variance; BBD: Box-Behnken
design; C.V: Coefficient of variation; MIC: Minimum inhibitory concentration; LC-ESI-MS: Liquid chro-
matography-electron spray ionization-mass spectroscopy

Introduction poverty and insufficient advanced medical resources.[9]

The deep eutectic solvents (DESs) came into view as
a new green substitute of traditional toxic organic sol-
vents and have been increasingly used in different
research areas.[1] They composed of a mixture of hydro-
gen bond acceptor and donor species having supramo-
lecular structure with melting point significantly lesser
than individual components.[2–4] These solvents have
similar physico-chemical properties of ionic liquids but
are accessible at low cost and biologically degradable.[5]
The DESs can be prepared easily without using harsh
organic solvents, which cause environmental pollution.
They have been attributed as environment-friendly sub-
stituents of conventional volatile organic solvents.
Additionally, they are employed for the extraction and
separation of bioactive ingredients (secondary metabo-
lites) from medicinal plants.[3] Various DESs, which
contain choline chloride with different hydrogen bond
donors, were screened for the extraction of natural pro-
ducts and proved efficient as compared to the organic
solvents for the extraction and separation of
anthocyanin[6] and phenolic compounds.[7,8]
About 65–80% population of world relies on thera-
peutic foliage for their prime health concern, due to
Nature has been a resource of remedial compounds for
thousands of years and huge number of advanced drugs
have been extracted and isolated from green
resources.[10] The phytochemicals of medicinal plants
have potential to combat against infectious and chronic
diseases.[11,12] It is imperative to investigate the signifi-
cance of widespread flora as they constitute traditional
and advanced therapeutic medicines. The extraction of
medicinally active components from plants and their
evaluation (quantitative and qualitative) is imperative
for exploration of new potent drugs. The choice of
solvent, extraction technique and process parameters
(time, temperature, solid to liquid ratio, etc.) are most
imperative factors for the separation of biologically
active components from plants during extraction
procedure.[13] The sonication method is considered
green extraction method due to short extraction time,
less solvent consumption; reduced energy and signifi-
cant yield of extracted ingredients. The bubbling and
cavitation phenomena during ultrasonic process
increase pressure and temperature which cause collapse
of the cavities near the plant materials and ultimately
break the cell wall. Hence, the solvent absorption into

CONTACT Muhammad Zahid rmzahid@uaf.edu.pk; zahid595@gmail.com Chemistry Department University of Agriculture, Faisalabad, Pakistan
Supplemental data for this article can be accessed on the publisher’s website.
© 2020 Taylor & Francis
2 Z. NASEEM ET AL.
plant material increases the mass transfer of phytochem-
icals into the solvent.[14,15]
The optimization of process parameters is done using
empirical and statistical approaches. In classical study,
the response is direct function of single variable but the
actual response is produced due to interaction among
diverse factors. On the other hand, statistical evaluation
due to interactive influence is more reliable than classi-
cal methods.[16] The response surface methodology
(RSM) facilitates to evaluate interactive effects of inde-
pendent variables on the response variables. Therefore,
RSM is supportive for building models, designing
experiment, to find the linear and interactive effects of
several parameters simultaneously to obtain optimum
conditions.[17]
Cymbopogon citratus Stapf belongs to the family of
Poaceae and universally known as lemongrass which is
extensively used in customary medication.[18–20] The
reported ethnomedicinal compounds of C. citratus are
phenolics and flavonoids which consist of quercetin, api-
ginin, luteolin, isoorientin 2ʹ-O-rhamnoside, and
kaempferol.[21] The C. citratus presents venerable remedy
for headache, cough, flu, malaria, pneumonia, and for
muscular disorder. The lemongrass is a good antiseptic
that smooths the process of detoxification of pancreas,
kidney, liver, gall bladder, and the digestive tract.[22]
The aim of present study was to investigate the effi-
ciency of DES over aqueous methanol for the better
extraction of phytochemicals. Choline chloride-glycerol-
based DES was prepared and used for the extraction of
phenolics/flavonoids from C. citrates using maceration as
well as ultrasonic-assisted extraction (UAE). The initial
optimization of process parameters, i.e., time, tempera-
ture, and solid to liquid ratio was carried out by conven-
tional study followed by statistical evaluation of
phytochemicals (TPC, TFC, and DPPH) through Box-
Bhenken design (BBD) by RSM. In vitro antibacterial
and antifungal activities of plant extracts were assessed
by measuring inhibition zone and minimum inhibition
concentration (MIC). Different bioactive components
have been characterized using LC-ESI-MS. The current
scientific study provides excellent alternative solvent for
the extraction and separation of bioactive compounds
with high yield from medicinal plants.
Material and methods
Collection and preservation of plant material
Cymbopogon citratus Stapf plant as a whole was har-
vested in mid-October, 2016 from New Botanical
Garden (NBG) of University of Agriculture, Faisalabad,
Pakistan. The valid identification was conceded by
Dr. Mansoor Hameed, Associate Professor of
Department of Botany, University of Agriculture,
Faisalabad, Pakistan. The plant material was cleaned
with tap water to remove the dirt particles and dried in
shade (25 ± 5 °C) approximately for 30 days. The dried
material was ground using mechanical grinder, sieved to
achieve the mesh size of 250 µm. The voucher sample
was kept in air tight container, assigned with number,
and stored at ambient temperature (25 ± 5 °C) for
further analysis.
Chemicals and reagents
Choline chloride (99%, Dae-Jung, Siheung, Korea), gly-
cerol (99.50%, Dae-Jung, Siheung, Korea), Folin-
Ciocalteu reagent (99.99%, Sigma-Aldrich, Darmstadt,
Germany), DPPH (99.50%, Sigma-Aldrich, Darmstadt,
Germany), aluminum chloride (99.99%, Sigma-Aldrich,
Darmstadt, Germany), sodium carbonate (99%,
Honeywell Riedel-deHaen, Seelze, Germany), potassium
acetate (Sigma-Aldrich, 99%, Darmstadt, Germany),
nutrient agar (99%, HiMedia, Pennsylvania, USA),
potato dextrose agar (99%, TM Media PR, London,
UK), sabarose dextrose broth (99%, AppliChem
GmbH, Darmstadt, Germany), resazurin (99% WRH
Int. Limited Harlow, UK).
Analytical reference standards of rutin, primulaverin,
sinapic acid, caffeic acid, quercetin, quercetin
3-O-glucuronide, chlorogenic acid, chrysoeriol 7-ruti-
noside, apigenin-6-C-glucoside, calycosin with more
than ≥98.00% purity were purchased from the Sigma
Aldrich. Stock solutions (1.0 mg/mL) were prepared in
methanol. Dilution of mixed standards was carried out
with acetonitrile/water (18/82, v/v). Working standard
solutions mixture (1.0 µg/mL) for validation were pre-
pared from stock solutions of particular analytical
standard.[23]
Synthesis of DES
The choline chloride and glycerol used for the prepara-
tion of DES, were oven dried at 60 °C for 24 h before use.
These individual components, i.e., choline chloride and
glycerol were mixed with mole ratios (1:2), and heated at
80 °C to get colorless, translucent, homogenous and
hydrogen-bonded supramolecular liquid.[3,24]
Hydrogen-bonded supramolecular structure of choline
chloride and glycerol is shown in Fig. 1.
Extraction of phytochemicals
In the maceration process, 2 g dried powder of
C. citratus was extracted with 20 mL of DES and 80%
 SEPARATION SCIENCE AND TECHNOLOGY 3

Figure 1. Hydrogen-bonded supramolecular network of choline chloride and glycerol (1:2) generated using Gauss View 5.

aq. MeOH[25] independently with magnetic stirring at weight of extracted product

Extraction yield ðmg=gÞ ¼
different levels of time, temperature, and solid to liquid weight of plant material
ratio. The maceration with continuous stirring was done
by magnetic stirrer with Teflon lined magnet bar in
a 100 mL Pyrex-glass flask. In UAE, 2 g dried material
was extracted with 20 mL of DES and 80% aq. MeOH,[26] Response surface methodology
kept in an ultrasonic bath (2.5 L 250 W DSA50-SK2 The parameters which influenced the extraction process
Digital Ultrasonic Cleaner) at different levels of sonica- of phenolics compounds were evaluated and optimized by
tion time, temperature, and solid to liquid ratio.[13] The Box-Behnken design.[28] The effects of independent vari-
extraction was performed with generation of 40 KHz ables like extraction time (A), temperature (B) and solid
frequency. The maceration was performed as classical to liquid ratio (C) were evaluated at three levels of varia-
method to compare the results of UAE. tion. The upper and lower levels of these independent
variables were evaluated with both solvent in preliminary
experiments during extraction yield determination. The
Centrifugation
seventeen experiments were conducted randomly to ana-
The mixture of extraction solvent and biomass were filtered lyze the significance of applied model against TPC, TFC,
by vacuum filtration to separate the extracts from residue and DPPH inhibition responses. The experimental data of
of plant biomass. The filtered extract of each sample (2 mL) dependent variables were fitted in second-order polyno-
was diluted 10 times with water and centrifuged at mial equation which is given in Eq. (1).
6500 rpm up to 60 min to separate bioactive compounds X3
from solvents. The bioactive compounds have been pre- Y ¼ β0 þ β 0 Xi
X3 i¼1 X
2 X3
cipitated and settled at the bottom and the solvents (DES þ β X 2
β X (1)
i¼1 ii i i¼1 j¼if1 ij iXj
and aqueous methanol) were separated as upper layer. The
solid material separated from bottom layer was dried in Y = Predicted response
desiccator until constant weight achieved. The dried solid βo = Coefficient for intercept
fractions of all extracts were kept at 4 °C for further βi = Linear coefficient
analysis.[27] βii = Quadratic coefficient
βij = Coefficient for interaction terms
The analysis of variance (ANOVA) was applied to
Extraction yield
examine the implication of statistical terms in the regres-
The yield of extracted phytochemicals is calculated after sion equation. The statistically non-significant (p ˃ 0.05)
obtaining the dry weight from extract terms were not considered in the model and the
4 Z. NASEEM ET AL.

Table 1. Experimental values and coded levels of independent modification.[34] The detailed procedure of antimicro-
variables used for BBD. bial activities can be seen in supplementary information.
Independent variables Factor levels
−1 0 1
Maceration Liquid chromatography-electrospray ionization
Time (hours) 6 9 12
Temperature (oC) 40 60 80 mass spectrometry
Solid/liq. (g/10 mL) 0.5 1.0 1.5
UAE The phenolics and flavonoids from the extracts (at opti-
Time (min.) 30 45 60
Temperature (oC) 50 60 70
mized conditions) were identified by liquid chromato-
Solid/liq. (g/10 mL) 0.5 1.0 1.5 graphy coupled with mass spectrometry (LC-ESI-MS,
8050 Shimadzu, Japan). The C18 column (2.1 mm ×
150 mm) with RI830 detector was used for the separa-
experimental data were justified only by significant (p ˂ tion. The methanol/water was used as mobile phase.
0.05) terms. Isocratic elution was executed with the flow rate of
The optimization of extraction time, temperature, 0.2 mL/min by subsequent run conditions; (i) 65% of
and solid to liquid ratio (independent variables) was methanol from 0 to 30 min, (ii) 55%, from 31 to 40 min
examined comprehensively by BBD for the extraction (iii) 35%, at 41–60 min. The sample (20 µL) was injected
of phytochemicals. The experimental and coded levels of and identification of the compounds was carried out
independent variables used for BBD are shown in under the conditions of negative and positive ion
Table 1 mode with 50–2000 m/z (mass range), 4 kV (electrical
potential), 325 °C (gas temperature) and 40 psi (nebuliz-
ing pressure).
Evaluation of phytochemicals
The evaluation of antioxidant activities was done using
Statistical analysis
the crude liquid sample and DES was used as blank to
nullify the solvent effect. The Folin-Ciocalteau’s reagent All experiments were performed in triplicates. The
method was used for the determination of total phenolic experimental results were expressed as mean ± standard
content (mg GAE/G) of plant extracts by deviation (n = 3). The levels of significance (P ˂0.05)
spectrophotometer.[29] The aluminum chloride colori- were evaluated using analysis of variance (ANOVA).
metric method with slight modification was used to Response surface methodology was performed using
analyze the total flavonoids content (mg QE/g) of plant Design Expert Software 7.0. The schematic diagram of
extract.[30] The free radical scavenging potential of plant methodology is given in supporting information
extracts was determined using DPPH radical (% (Figure S1).
inhibition).[31,32] The detail of the experimental methods
for the evaluation of phytochemicals activities are given
in the supplementary information. Results and discussion
Extraction yield

Antimicrobial activities The extraction of phytochemical constituents from

Antibacterial activity
The antibacterial potential of C. citratus extracts against
S. aureus (Gram-positive) and E. coli (Gram-negative)
bacterial strains was investigated. The antibacterial
activity of plant extracts was evaluated by well diffusion
assay. Minimum inhibitory concentration (MIC) for
each extract was determined by Broth micro-dilution
method.[33]
Antifungal activity
Two fungal strains F. solani and A. niger were used to
test the antifungal potential of C. citratus extracts. MIC
of plant extracts against fungal strains was determined
by following the already reported protocol with some
C. citratus was carried out using ChCl:Gly DES and
aqueous methanol. Maceration and UAE were applied
for the extraction purpose. The optimization of process
parameters (time, temperature, and solid to liquid ratio)
was done. The yield of phytochemicals significantly
depends on the extraction methods and solvent/media
used.
Effect of time
The optimum extraction time in maceration and UAE
would be very effective for the maximum extraction of
phytochemicals. The preliminary study was performed
for 3 to 24 h at 60 °C with 1 g/10 mL solid to liquid ratio
during maceration. The highest extraction yield of
297 mg/g (in 9 h) and 193 mg/g (in 15 h) were observed

Figure 2. Effect of time (a & d), temperature (b & e) and solid to liquid ratio (c & f) on extraction yield during maceration (a, b & c) and
UAE (d, e & f).
for ChCl:Gly and aq. MeOH, respectively. No further
change was observed up to 24 h as shown in Fig. 2(a).
The Fick’s second law of diffusion describes that the
solute in the matrix of plant equilibrate with the bulk
of solvent around it. The change in time of solvent-
solute equilibrium and concentration of phenolics
depends on the diffusion rate which is based on the
solubility of phytochemicals in solvent.[35] In UAE, the
extraction was carried out for 15–60 min and results are
as shown in Fig. 2(d). The ChCl:Gly extract quantity
increased (229 mg/g) up to 60 min while the optimized
time of aq. MeOH was 45 min with 126 mg/g yield. The
cavitation effects increased with the increase of time in
UAE, which ultimately enhanced the mass transfer and
rate of extraction.[36]
Effect of temperature
The temperature is also a significant factor to assist the
extraction process. In maceration, the extraction was
carried out at 30–70°C. It was evaluated that the yield
was increased with the rise in temperature up to 60°C
(294 mg/g) and then remained constant (295 mg/g) at 70
°C. In case of aq. MeOH, the optimized temperature 60 °
C with 230 mg/g yield was observed as shown in Fig. 2
(b). In UAE, the temperature effect was studied at 40–70
°C and obtained temperature was 60 °C for the both
SEPARATION SCIENCE AND TECHNOLOGY 5
ChCl:Gly and aq. MeOH with extraction yield of
250 mg/g and 145 mg/g, respectively, as shown in Fig.
2(e). With the increase in temperature, the diffusion of
phytochemicals increased while the viscosity of the sol-
vent decreased. As reported in literature, the viscosity of
ChCl:Gly is decreased from 281 cP to 79 cP for the rise
in temperature from 298 K to 353 K.[37] Hence, the
solubility of phytochemicals increased with the rise in
temperature during extraction. However, the decompo-
sition of phenolics limits the increase in temperature. It
was observed that moderate temperature provided posi-
tive effects on the extraction of phytochemicals yield.
Effect of solid to liquid ratio
The yield largely depends on the solid to liquid ratio so
its optimization is also necessary for the maximum
extraction. The extraction by maceration and UAE was
done using 0.5–2 g/10 mL solid to liquid ratio with
respective optimized conditions of time and tempera-
ture. In maceration, as shown in Fig. 2(c) the optimum
yields of 299 mg/g was recorded with ChCl:Gly solvent
at 1.0 g/10 mL and then continuous decrease was
observe up to 2.0 g/10 mL while the aq. MeOH showed
220 mg/g yield with 1.5 g/10 mL solid to liquid ratio then
became constant. In UAE, both the ChCl:Gly and the aq.
MeOH provided highest yield at 1.5 g/10 mL which
6 Z. NASEEM ET AL.
remained almost constant up to 2.0 g/10 mL as shown in
Fig. 2(f). The rate of diffusion of solute molecules into
solvent was directly influenced by the solid to liquid
ratio. A small solid to liquid ratio can dissolve bioactive
ingredients with more efficacy, leading to an enhance
extraction yield.[38]
It has been observed that the extraction efficacy is
primarily based on the dissolving ability of solvents.
Phenolic compounds are generally polar and solvent
appears to play a significant role in their extraction so
that polar solvents tend to contain more of these com-
ponents compared to the less polar or nonpolar
solvents.[39] The DES have strong hydrogen bonding,
which ultimately improved the extraction efficiency by
increasing the dissolution level.[3] The prepared DES
showed enhanced extraction yield compared with aqu-
eous methanol.
Response surface methodology
The optimization of extraction time, temperature, and
solid to liquid ratio (independent variables) was exam-
ined comprehensively using BBD.[40] Three responses,
TPC, TFC, and % inhibition of DPPH were evaluated by
applying the second-order polynomial equation. The
fairly high regression coefficient (R2) values for TPC,
TFC, and DPPH with least standard deviations during
maceration as well as UAE described applied regression
model was well fitted. The validation of quadratic model
with small C.V (less than 5% in all responses) explained
the better reliability and good precision of data. The lack
of fit with more than 0.05 p-values also illustrated the
validity of applied quadratic model in both extraction
methods.[41] Sum of squares (SS) relate to the total
variance of the observations. The sample variance is
also referred to as a mean square (MS) because it is
obtained by dividing the sum of squares by the respec-
tive degrees of freedom (df). In Tables 3 and 5, the SS,
MS, and df values of analysis of variance are given. The
interaction between independent variable and their
mutual influence on the response values is depicted by
3D response surfaces. The contours represent the inter-
actions between independent variables and responses,
keeping other independent variable at fixed level. The
independent variables, i.e., time (A), temperature (B)
and solid to liquid ratio (C) with their combined influ-
ences (AB, BC, and AC) on the extraction of phenolic
and flavonoids contents and % inhibition of DPPH
during maceration were depicted in response surfaces
as shown in Fig. 3. It is observed that time, temperature,
solid to liquid ratio and their interactions have positive
effects upto some extent and then decline started. The
medium points showed maximum yield of TPC, TFC,
and antioxidant activity. In UAE, the interactions
between independent variables are illustrated in contour
surfaces as shown in Fig. 4. The upper and lower levels
of time, temperature, and solid to liquid ratio did not
show the valuable results. The medium levels of process
variables preferential for effective extraction.
The inconsistency between the predicted and the
experimental values is identified as residual and it
imparts momentous role to judge the significance of
applied model. Therefore, if the statistical model is well
fitted, its residuals graphs present a behavior that por-
trays a normal distribution.[42] The graphs of normal %
probability versus studentized residuals of all three pro-
cess parameters of both extraction methods are pre-
sented in Figure S2-S4 (supplementary information).
The perturbation plots demonstrate the deviation of
the factorial level from the adjusted reference point of
all variables. It can be seen from the perturbation plots
that all independent variables, e.g. time (A), temperature
(B) solid to liquid ratio (C) act as the controlling factors
for the utmost yield. The perturbation plots are given in
supplementary information Figure S5-S7.
The extraction through maceration process was
investigated at different conditions of time (6–12 h),
temperature (40–80 °C) and solid to liquid ratio
(0.5–1.5 g/10 mL), which were chosen on the basis
of preliminary experiments. It was found that the
highest values of TPC (95 ± 2.75 mg GAE/g), TFC
(86 ± 3.0 mg QE/g), and DPPH (89%±2.00) were
achieved at 60 °C after 9 h of maceration with 1 g/
10 mL solid to liquid ratio. The experimental values
were in good agreement with those of predicted
(shown in Table 2), thus indicating adequacy of the
developed model. It is considered that high tempera-
ture leads to decomposition of phenolic contents and
more extraction time leads to the unwanted oxidation
of these compounds. The higher solid to liquid ratio
reduced the quantitative yield because less amount of
solvent is available for solubilization.[43] Consequently,
the moderate conditions are necessary for maximum
and safe recovery of these compounds. The ANOVA
of these experiments, following quadratic model equa-
tions in term of coded variables, showed the effect of
operating parameters for all three responses is given in
Table 3.
For the UAE, the extraction was conducted at three
different levels for each of time (30–60 min), tempera-
ture (50–70 °C), and solid to liquid ratio (1.0–2.0 g/
10 mL). The highest values of TPC (135 ± 2.50 mg
GAE/g), TFC (93 ± 2.50 mg QE/g), and DPPH
(94 ± 1.75%) were observed in 45 min under 60 °C
with 1.5 g/10 mL solid to liquid ratio. The experimental
values of TPC, TFC, and DPPH showed good agreement

Figure 3. The 3D surfaces for the effects of process parameters on the yield of TPC (a, b, c), TFC (d, e, f), and antioxidant activity in terms
of DPPH percent inhibition (g, h, i) by maceration.
with predicted values (Table 4). It was observed that
DPPH activity was maximum when the TPC and TFC
contents were the highest in extracts. This shows posi-
tive correlation between TPC, TFC, and DPPH activity.
The ability of plant extract to scavenge DPPH also
reflects its potential to inhibit the formation of free
radical. This implies that the C. citratus extract is useful
for treating radical-related pathological damage.[44] The
structural composition and hydrogen bonding of DES as
well as sonication process played a major role to extract
the higher content of phenolics and flavonoids.[45] It has
been observed from the results of RSM treatment that
TPC TFC and DPPH showed utmost quantitative yield
and activity at the same conditions which has shown
maximum crude yield (in preliminary studies). The
ANOVA (for experiments conducted through UAE)
SEPARATION SCIENCE AND TECHNOLOGY 7
and quadratic model equations in terms of coded vari-
ables for all three responses are given Table 5. It is
concluded that model linear terms (A, B, C), interaction
terms (AB, AC, BC), and quadratic terms (A2, B2, C2) are
influencing and show level of significance with p value
<.05 for all response variables.
Antimicrobial activities
Phytochemicals constituents have precious curative
agents to treat the human being diseases with supple-
mentary efficacies having minimum or no side
effects.[46] A massive amount of scientific study proved
that plants bioactive constituents have aptitude to pro-
duce antimicrobial agents as defense mechanism to
guard them against microorganisms and biotic stress.[47]
8 Z. NASEEM ET AL.

Figure 4. The 3D surfaces for the effects of process parameters on the yield of TPC (a, b, c), TFC (d, e, f), and antioxidant activity in terms
of DPPH percent inhibition (g, h, i) by UAE.

Table 2. The experimental and predicted values of TPC, TFC, and DPPH of ChCl:Gly extracts of C. citratus by maceration.
Time (h) Temperature (oC) Solid/liq. (g/10 mL) TPC (mg GAE/g) TFC (mg QE/g) DDPH (% inhibition)
Sr. No A B C Exp. Pred. Exp. Pred. Exp. Pred.
1 12.00 60.00 1.50 88 ± 2.50 89 68 ± 2.50 68 53 ± 1.25 54
2 12.00 80.00 1.00 51 ± 1.25 51 40 ± 0.50 39 57 ± 1.25 56
3 9.00 60.00 1.00 95 ± 2.75 94 73 ± 2.50 73 89 ± 2.00 88
4 9.00 80.00 0.50 33 ± 0.75 33 18 ± 0.25 17 45 ± 1.00 44
5 6.00 80.00 1.00 71 ± 1.50 72 58 ± 1.00 59 50 ± 1.25 51
6 9.00 40.00 0.50 43 ± 1.00 44 23 ± 0.50 23 45 ± 1.00 45
7 6.00 60.00 1.50 74 ± 2.00 75 54 ± 1.00 53 74 ± 2.00 73
8 6.00 60.00 0.50 54 ± 1.25 53 32 ± 0.50 31 50 ± 1.25 49
9 9.00 80.00 1.50 59 ± 1.50 57 32 ± 0.50 33 46 ± 1.00 45
10 9.00 60.00 1.00 92 ± 2.50 94 73 ± 1.25 73 89 ± 2.00 88
11 9.00 60.00 1.00 95 ± 2.50 94 73 ± 1.25 73 87 ± 2.50 88
12 12.00 40.00 1.00 95 ± 2.50 96 86 ± 3.00 87 56 ± 1.25 54
13 9.00 60.00 1.00 95 ± 2.50 94 74 ± 1.25 73 87 ± 2.50 88
14 9.00 60.00 1.00 94 ± 2.50 94 73 ± 1.25 73 88 ± 2.50 88
15 9.00 40.00 1.50 55 ± 1.25 54 49 ± 1.50 50 60 ± 1.50 59
16 12.00 60.00 0.50 76 ± 1.75 75 49 ± 1.50 49 59 ± 1.50 60
17 6.00 40.00 1.00 37 ± 0.75 36 34 ± 0.75 34 69 ± 1.50 69
Table 3. ANOVA of TPC TFC and DPPH of C. citratus extract of ChCl:Gly by maceration.
TPC TFC DPPH
Source SS df MS F-value p-value SS df MS F-value p-value SS df MS F-value p-value
Model 8056.20 9 895.13 454.05 0.0001 6902.19 9 766.91 2618.72 0.0001 4695.94 9 521.77 332.04 0.0001
A-Time 684.50 1 684.50 347.21 0.0001 528.13 1 528.13 1803.35 0.0001 40.50 1 40.50 25.77 0.0014
B-Temp. 32.00 1 32.00 16.23 0.0050 231.13 1 231.13 789.21 0.0001 128.00 1 128.00 81.45 0.0001
C-Solid/liq. 612.50 1 612.50 310.69 0.0001 840.50 1 840.50 2870.00 0.0001 144.50 1 144.50 91.95 0.0001
AB 1521.00 1 1521.00 771.52 0.0001 1225.00 1 1225.00 4182.93 0.0001 100.00 1 100.00 63.64 0.0001
AC 16.00 1 16.00 8.12 0.0247 2.25 1 2.25 7.68 0.0276 225.00 1 225.00 143.18 0.0001
BC 49.00 1 49.00 24.86 0.0016 30.25 1 30.25 103.29 0.0001 49.00 1 49.00 31.18 0.0008
A2 28.46 1 28.46 14.44 0.0067 1.78 1 1.78 6.07 0.0432 421.05 1 421.05 267.94 0.0001
B2 3324.67 1 3324.67 1686.43 0.0001 1576.52 1 1576.52 5383.22 0.0001 1684.21 1 1684.21 1071.77 0.0001
C2 1456.67 1 1456.67 738.89 0.0001 2246.78 1 2246.78 7671.93 0.0001 1520.00 1 1520.00 967.27 0.0001
Residual 7.00 7 1.97 2.05 7 0.29 11.00 7 1.57
Lack of Fit 6.80 3 2.33 1.37 0.3717 1.25 3 0.42 2.08 0.5413 7.00 3 2.33 2.33 0.2155
Pure Error 8070.00 4 1.70 0.80 4 0.20 4.00 4 1.00
Cor Total 16 6904.24 16 4706.94 16
Std. Dev. 1.40 R-Squared 0.9983 Std. Dev. 1.54 R-Squared 0.9997 Std. Dev. R-Squared 0.9977
1.25
Mean 71.00 Adj. R-Squared 0.9961 Mean 53.53 Adj. R-Squared 0.9993 Mean Adj. R-Squared 0.9947
64.94

SEPARATION SCIENCE AND TECHNOLOGY

C.V. % 1.98 Pred R-Squared 0.9848 C.V. % 1.01 Pred R-Squared 0.9969 C.V. % Pred R-Squared 0.9749
1.93
TPC = – 462.2500 + 29.11667A + 10.90500B + 157.300 C – 0.3250AB – 1.3334AC + 0.3500BC – 0.28889A2 – 0.070250B2 – 74.400C2
TFC = – 394.7500 + 19.40833A + 8.43625B + 226.3000 C – 0.29167AB – 0.500AC – 0.27500BC + 0.072222A2 – 0.048375B2 – 92.4000C2
DPPH = – 268.750 + 19.2500A + 5.4000B + 226.5000 C – 0.083333AB – 5.00AC – 0.3500BC – 1.1111A2 – 0.05000B2 – 76.000C2

9
 10 Z. NASEEM ET AL.

Table 4. The experimental and predicted values of TPC, TFC, and of DPPH of ChCl:Gly extracts of C. citratus by UAE.
Time (min) Temperature (oC) Solid/liq. (g/10 mL) TPC (mg GAE/g) TFC (mg QE/g) DDPH (% inhibition)
Sr. No A B C Exp. Pred. Exp. Pred. Exp. Pred.
1 60.00 50.00 1.50 89 ± 1.50 89 76 ± 1.50 78 76 ± 1.70 75
2 45.00 60.00 1.50 135 ± 2.50 133 89 ± 2.50 91 94 ± 1.75 93
3 45.00 60.00 1.50 133 ± 2.50 133 91 ± 2.50 91 92 ± 1.80 93
4 60.00 60.00 2.00 76 ± 2.50 73 65 ± 2.00 62 64 ± 1.50 64
5 45.00 70.00 1.00 63 ± 2.75 60 40 ± 0.75 39 50 ± 1.00 49
6 30.00 60.00 1.00 82 ± 2.25 84 55 ± 1.00 58 58 ± 1.25 58
7 45.00 60.00 1.50 134 ± 2.75 133 90 ± 2.25 91 90 ± 2.00 93
8 45.00 60.00 1.50 131 ± 2.75 133 93 ± 2.50 91 92 ± 1.80 93
9 45.00 70.00 2.00 61 ± 1.30 62 45 ± 1.00 46 58 ± 1.25 58
10 60.00 60.00 1.00 67 ± 1.45 68 61 ± 1.75 59 64 ± 1.50 65
11 30.00 50.00 1.50 86 ± 1.40 85 63 ± 1.50 60 79 ± 1.80 79
12 30.00 60.00 2.00 68 ± 1.30 66 43 ± 0.80 44 67 ± 1.50 65
13 45.00 50.00 1.00 77 ± 1.70 75 61 ± 1.75 60 63 ± 1.50 62
14 45.00 50.00 2.00 60 ± 2.35 62 42 ± 0.80 43 59 ± 1.25 60
15 45.00 60.00 1.50 132 ± 2.25 133 93 ± 2.50 91 94 ± 1.75 93
16 30.00 70.00 1.50 86 ± 1.50 86 62 ± 2.00 59 64 ± 1.50 65
17 60.00 70.00 1.50 71 ± 1.00 72 59 ± 2.00 61 75 ± 1.70 74

Antibacterial activity F. solani and A. niger, respectively. The results obtained

Two bacterial strains, i.e., S. aureus and E. coli were used to
check the potential of extracts against these microorgan-
isms. The antibacterial activities of plant extracts (obtained
at optimized conditions) by maceration (9 h, 60 °C, and
1 g/10 mL) and UAE (45 min, 60 °C, and 1.5 g/10 mL) were
evaluated. The antibacterial activities were assessed quan-
titatively by measuring the inhibition zones. The MIC by
micro-dilution method of all extracts were also deter-
mined, which showed the inhibition zone.[48] The ChCl:
Gly extracts have shown more potential to extinct both
Gram-positive (S. aureus) and Gram-negative (E. coli) bac-
terium than aq. MeOH extracts in maceration and UAE.
The ChCl:Gly extract obtained by UAE showed larger zone
of inhibition (40.5 ± 1.5 mm and 39.5 ± 1.4 mm) with
minimum MIC (12.5 ± 1.5 µg/mL and 25 ± 2.0 µg/mL)
against S. aureus and E. coli, respectively. The plant extracts
showed the antibacterial potential but less than that of
standard Rifampicin. The results are shown in Table 6.
Antifungal activity
Two fungal strains, i.e., F. solani and A. niger were used
to assess the potential of C. citratus against these fungal
agents. The antifungal activity of extracts obtained
through maceration and UAE (at optimized conditions
in both cases) was also quantitatively determined by
measuring the inhibition zones as well as the MIC. The
ChCl:Gly extracts proved more potential to destroy
F. solani and A. niger fungi as compared to aq. MeOH
extracts in both maceration and UAE. In comparison
with aq. MeOH extracts, the ChCl:Gly extracts obtained
by applying UAE showed larger inhibition zone
(41 ± 1.4 mm and 34 ± 1.4 mm) with MIC
(12.5 ± 1.0 µg/mL and 12.5 ± 1.5 µg/mL) against
for all extracts along with standard Terbinafine are
shown in Table 6.
Identification of bioactive compounds through
LC-ESI-MS
The mass spectral identification of phenolics and flavo-
noids structures is widely carried out with electron spray
ionization (ESI) technique. This ionization technique has
been applied to identify phenolics flavonoids. Complex
fragmentation may occur with ESI due to the broad
spread of internal energy containing the initially pro-
duced M+• ions, which may hide or suppress the M+•
ions and significant primary fragment ions. The chemical
structures of identified compounds are given in Figure S8.
The identified compounds in this study were Primiverin/
Primulaverin, sinapic acid, rutin, quercetin
3-O-glucuronide, chrysoeriol 7-rutinoside, apigenin-
6-C-glucoside, and calycosin. The Primiverin/
Primulaverin was identified with Rt 8.1 min and detected
as [M + H]+ ion at m/z 475.2. The sinapic acid with Rt
8.15 min was identified as [M + H]+ ion at m/z 224.1. The
rutin with Rt 24.57 min was identified as [M + H]+ ion
at m/z 611.4. Quercetin 3-O-glucuronide was identified as
[M + H]+ ion at m/z 478.1 with Rt of 20.28 min and
Chrysoeriol 7-rutinoside was identified as [M + H]− ion
at m/z 608.1 with 45.324 min. Apigenin-6-C-glucoside
with Rt of 18.11 min was identified at m/z 342.2.
Calycosin with Rt of 3.43 min was identified as
[M + H]+ ion at 285.1. The LC-ESI-MS spectra of
C. citratus are shown in supplementary Figures S9–S15.
The phenolics and flavonoids extracted from C. citratus
are given in Table S1.
Table 5. ANOVA of TPC TFC and DPPH of C. citratus extract of ChCl:Gly by UAE.
TPC TFC DPPH
Source SS df MS F-value p-value SS df MS F-value p-value SS df MS F-value p-value
Model 13494.3 9 1499.37 257.56 0.0001 5664.58 9 629.40 84.81 0.0001 3495.83 9 388.43 195.08 0.0001
A-Time 45.13 1 45.13 7.75 0.0271 180.50 1 180.50 24.32 0.0017 13.78 1 13.78 6.92 0.0339
B-Temp. 120.13 1 120.13 20.63 0.0027 162.00 1 162.00 21.83 0.0023 116.28 1 116.28 58.40 0.0001
C-Solid/liq. 72.00 1 72.00 12.37 0.0098 55.13 1 55.13 7.43 0.0295 21.13 1 21.13 10.61 0.0139
AB 81.00 1 81.00 13.91 0.0074 60.06 1 60.06 8.09 0.0249 45.56 1 45.56 22.88 0.0020
AC 132.25 1 132.25 22.72 0.0020 68.06 1 68.06 9.17 0.0192 20.25 1 20.25 10.17 0.0153
BC 56.25 1 56.25 9.66 0.0171 150.06 1 150.06 20.22 0.0028 36.00 1 36.00 18.08 0.0038
A2 1856.84 1 1856.84 318.97 0.0001 321.45 1 321.45 43.31 0.0003 188.31 1 188.31 94.58 0.0001
B2 3541.05 1 3541.05 608.28 0.0001 1287.63 1 1287.63 173.50 0.0001 651.33 1 651.33 327.13 0.0001
C2 6322.37 1 6322.37 1086.05 0.0001 2954.05 1 2954.05 398.04 0.0001 2143.44 1 2143.44 1076.52 0.0001
Residual 40.75 7 5.82 51.95 7 7.42 13.94 7 1.99
Lack of Fit 30.75 3 10.25 4.10 0.1032 40.75 3 13.58 4.85 0.0806 6.44 3 2.15 1.14 0.4327
Pure Error 10.00 4 2.50 11.20 4 2.80 7.50 4 1.87
Cor Total 13535.0 16 5716.53 16 3509.76 16
TPC TFC DPPH
Std. Dev. 2.41 R-Squared 0.9970 Std. Dev. 2.72 R-Squared 0.9909 Std. Dev. 1.41 R-Squared 0.9960
Mean 91.24 Adj. R-Squared 0.9931 Mean 66.29 Adj. R-Squared 0.9792 Mean 72.88 Adj. R-Squared 0.9909
C.V. % 2.64 Pred R-Squared 0.9625 C.V. % 4.11 Pred R-Squared 0.8829 C.V. % 1.94 Pred R-Squared 0.9673
TPC = – 1371.1250 + 8.89167A + 34.6375B + 379.500C – 0.0300AB + 0.76667AC + 0.7500BC – 0.09333A2 – 0.2900B2 – 155.00C2
TFC = – 757.2250 + 4.53667A + 19.8600B + 214.3500 C – 0.025833AB + 0.5500AC + 1.22500BC – 0.038833A2 – 0.17487B2 – 105.9500C2
DPPH = – 509.93750 + 1.86250A + 12.63125B + 251.500 C + 0.0225AB – 0.300AC + 0.6000BC – 0.029722A2 – 0.12437B2 – 90.2500 C2

Table 6. Antibacterial and antifungal activities of C. citratus extracts obtained at optimized conditions.
Antibacterial activity
Staphylococcus aureus Escherichia coli

SEPARATION SCIENCE AND TECHNOLOGY

Inhibition zone (mm) MIC (µg/mL) Inhibition zone (mm) MIC (µg/mL)
ChCl:Gly MeOH:H2O ChCl:Gly MeOH:H2O ChCl:Gly MeOH:H2O ChCl:Gly MeOH:H2O
Maceration 34.5 ± 1.5 20 ± 1.0 25 ± 1.5 50 ± 2.5 33.5 ± 0.8 12.5 ± 0.5 12.5 ± 1.5 50 ± 2.0
UAE 40.5 ± 1.5 25.0 ± 1.0 12.5 ± 1.5 25 ± 2.0 39.5 ± 1.4 27 ± 0.8 25 ± 2.0 25 ± 2.5
Rifampicin 65 ± 2.4 12.5 ± 0.9 61 ± 2.1 12.5 ± 0.9
Antifungal activity
Fusarium solani Aspergillus niger
Maceration 21 ± 1.4 17 ± 0.4 12.5 ± 1.5 25 ± 1.5 29 ± 0.8 23 ± 0.5 6.3 ± 1.0 25 ± 1.5
UAE 41 ± 1.4 20 ± 0.4 12.5 ± 1.0 50 ± 2.5 34 ± 1.4 31 ± 0.8 12.5 ± 1.5 25 ± 2.0
Terbinafine 55 ± 4.5 12.5 ± 0.9 55 ± 4.5 6.3 ± 0.9

11
12 Z. NASEEM ET AL.
Conclusion
It is concluded from this study that choline chloride-
glycerol binary solvent proved better alternative for the
extraction of phytochemicals from C. citratus compared
with aqueous methanol. RSM with three factors at three
levels of BBD proved to be a useful statistical method for
the optimization of process parameters with <0.05 level of
significance. The results of ANOVA also indicated that
the applied model adequately represented the actual rela-
tionship between the independent variables and their
interactions. The p-values <0.0001 indicated that the
quadratic model was highly significant and accurate.
Ultrasound-assisted extraction was a beneficial extraction
technique than maceration due to reduced extraction
time and this technique is environment-friendly and sus-
tains the quality of extracted bioactive ingredients. The
extracts obtained by DES using UAE also showed super-
ior antioxidant and antimicrobial activities as compared
to maceration. The ChCl:Gly can be used as a better
substitute of organic solvents for the efficient extraction
of phytochemical constituents from medicinal plants.
Novelty statement
In present work, innovative hydrogen-bonded supramolecular
deep eutectic solvent (DES) was synthesized by combining cho-
line chloride with glycerol. This solvent was first time used for the
extraction of bioactive contents of Cymbopogon citratus using
maceration and ultrasound-assisted extraction methods. This
non-evaporative solvent provides significant extraction yield as
compared to conventional aqueous methanol. The extensive
hydrogen bonding play a vital role for the maximum extraction
of hydrogen-bonded biological molecules. The phytochemicals
extraction has been carried out through conventional (macera-
tion) as well as energy efficient (ultrasonic-assisted) methodolo-
gies followed by the biological activities. The ultra-sonic-assisted
extraction and optimization of process parameters have not been
studied for Cymbopogon citratus in DES. The prepared green
solvent showed much higher yield as well as biological activities
than conventional organic solvent. Additionally, the choline
chloride-based DESs are cheap, easy to prepare, and environment
friendly. The eutectic-based solvents would provide green and
efficient alternative of environmentally detrimental of organic
solvents.
Acknowledgements
This study was accomplished with partial financial support of
HEC through Access to Scientific Instrumentation Program
(ASIP) under grant No. 20-2(8)/ASIP/R&D/HEC/17/00038.
Disclosure statement
All the authors declared no potential conflict of interest.
Funding
This work was supported by the Higher Education
Commission, Pakistan [20-2(8)/ASIP/R&D/HEC/17/00038.].
References
[1] Shawky, E.; Takla, S. S.; Hammoda, H. M.;
Darwish, F. A. Evaluation of the Influence of Green
Extraction Solvents on the Cytotoxic Activities of
Crinum (Amaryllidaeae) Alkaloid Extracts Using
In-vitro-in-silico Approach. J. Ethnopharmacol. 2018,
227, 139–149. DOI: 10.1016/j.jep.2018.08.040.
[2] Smith, E. L.; Abbott, A. P.; Ryder, K. S. Deep Eutectic
Solvents (Dess) and Their Applications. Chem. Rev.
(Washington, DC, U. S.). 2014, 114, 11060–11082.
DOI: 10.1021/cr300162p.
[3] Dai, Y.; van Spronsen, J.; Witkamp, G.-J.; Verpoorte, R.;
Choi, Y. H. Natural Deep Eutectic Solvents as New
Potential Media for Green Technology. Anal. Chim.
Acta. 2013, 766, 61–68. DOI: 10.1016/j.aca.2012.12.019.
[4] Li, Y.; Ali, M. C.; Yang, Q.; Zhang, Z.; Bao, Z.; Su, B.;
Xing, H.; Ren, Q. Hybrid Deep Eutectic Solvents with
Flexible Hydrogen-Bonded Supramolecular Networks
for Highly Efficient Uptake of NH3. ChemSusChem.
2017, 10, 3368–3377. DOI: 10.1002/cssc.201701135.
[5] Carriazo, D.; Serrano, M. C.; Gutiérrez, M. C.;
Ferrer, M. L.; Del Monte, F. Deep-eutectic Solvents
Playing Multiple Roles in the Synthesis of Polymers
and Related Materials. Chem. Soc. Rev. 2012, 41,
4996–5014. DOI: 10.1039/c2cs15353j.
[6] Jeong, K. M.; Zhao, J.; Jin, Y.; Heo, S. R.; Han, S. Y.;
Yoo, D. E.; Lee, J. Highly Efficient Extraction of
Anthocyanins from Grape Skin Using Deep Eutectic
Solvents as Green and Tunable Media. Arch.
Pharmacal Res. 2015, 38, 2143–2152. DOI: 10.1007/
s12272-015-0678-4.
[7] Wei, Z.; Qi, X.; Li, T.; Luo, M.; Wang, W.; Zu, Y.; Fu, Y.
Application of Natural Deep Eutectic Solvents for
Extraction and Determination of Phenolics in Cajanus
Cajan Leaves by Ultra Performance Liquid
Chromatography. Sep. Purif. Technol. 2015, 149,
237–244. DOI: 10.1016/j.seppur.2015.05.015.
[8] Barbieri, J. B.; Goltz, C.; Cavalheiro, F. B.; Toci, A. T.;
Igarashi-Mafra, L.; Mafra, M. R. Deep Eutectic Solvents
Applied in the Extraction and Stabilization of Rosemary
(Rosmarinus Officinalis L.) Phenolic Compounds. Ind.
Crops Prod. 2020, 144, 112049. DOI: 10.1016/j.
indcrop.2019.112049.
[9] Calixto, J. B. Twenty-five Years of Research on
Medicinal Plants in Latin America: A Personal View.
J. Ethnopharmacol. 2005, 100, 131–134. DOI: 10.1016/j.
jep.2005.06.004.
[10] Adebooye, O. C.; Alashi, A. M.; Aluko, R. E. A Brief
Review on Emerging Trends in Global Polyphenol
Research. J. Food Biochem. 2018, 42, e12519. DOI:
10.1111/jfbc.12519.
[11] Sharma, A.; Del Carmen Flores-Vallejo, R.; Cardoso-
Taketa, A.; Villarreal, M. L. Antibacterial Activities of
Medicinal Plants Used in Mexican Traditional
Medicine. J. Ethnopharmacol. 2017, 208, 264–329.
DOI: 10.1016/j.jep.2016.04.045.

[12] Venditti, A.; Frezza, C.; Campanelli, C.; Foddai, S.;
Bianco, A.; Serafini, M. Phytochemical Analysis of the
Ethanolic Extract of Agathis Robusta (C. Moore Ex
F. Muell.) FM Bailey. Nat. Prod. Res. 2017, 31,
1604–1611. DOI: 10.1080/14786419.2017.1288625.
[13] Dranca, F.; Oroian, M. Optimization of
Ultrasound-assisted Extraction of Total Monomeric
Anthocyanin (TMA) and Total Phenolic Content
(TPC) from Eggplant (Solanum Melongena L.) Peel.
Ultrason. Sonochem. 2016, 31, 637–646. DOI: 10.1016/
j.ultsonch.2015.11.008.
[14] Chanioti, S.; Tzia, C. Extraction of Phenolic
Compounds from Olive Pomace by Using Natural
Deep Eutectic Solvents and Innovative Extraction
Techniques. Innovative Food Sci. Emerg. 2018, 48,
228–239. DOI: 10.1016/j.ifset.2018.07.001.
[15] Mazza, K. E.; Santiago, M. C.; Do Nascimento, L. S.;
Godoy, R. L.; Souza, E. F.; Brígida, A. I. S.;
Borguini, R. G.; Tonon, R. V. Syrah Grape Skin
Valorisation Using Ultrasound-assisted Extraction:
Phenolic Compounds Recovery, Antioxidant Capacity
and Phenolic Profile. Int. J. Food Sci. Technol. 2019, 54,
641–650. DOI: 10.1111/ijfs.13883.
[16] Liyana-Pathirana, C.; Shahidi, F. Optimization of
Extraction of Phenolic Compounds from Wheat Using
Response Surface Methodology. Food Chem. 2005, 93,
47–56. DOI: 10.1016/j.foodchem.2004.08.050.
[17] Danmaliki, G. I.; Saleh, T. A.; Shamsuddeen, A. A.
Response Surface Methodology Optimization of
Adsorptive Desulfurization on Nickel/activated
Carbon. Chem. Eng. J. (Lausanne). 2017, 313,
993–1003. DOI: 10.1016/j.cej.2016.10.141.
[18] Costa, G.; Ferreira, J. P.; Vitorino, C.; Pina, M. E.;
Sousa, J. J.; Figueiredo, I. V.; Batista, M. T.
Polyphenols from Cymbopogon Citratus Leaves as
Topical Anti-inflammatory Agents. J. Ethnopharmacol.
2016, 178, 222–228. DOI: 10.1016/j.jep.2015.12.016.
[19] Marongiu, B.; Piras, A.; Porcedda, S.; Tuveri, E.
Comparative Analysis of the Oil and Supercritical
CO2 Extract of Cymbopogon Citratus Stapf. Nat.
Prod. Res. 2006, 20, 455–459. DOI: 10.1080/
14786410500277837.
[20] de Almeida Costa, C. A. R.; Kohn, D. O.; de Lima, V. M.;
Gargano, A. C.; Flório, J. C.; Costa, M. The GABAergic
System Contributes to the Anxiolytic-like Effect of
Essential Oil from Cymbopogon Citratus
(Lemongrass). J. Ethnopharmacol. 2011, 137, 828–836.
DOI: 10.1016/j.jep.2011.07.003.
[21] Shah, G.; Shri, R.; Panchal, V.; Sharma, N.; Singh, B.;
Mann, A. Scientific Basis for the Therapeutic Use of
Cymbopogon Citratus, Stapf (Lemon Grass). J. Adv.
Pharm. Tech. Res. 2011, 2, 3. DOI: 10.4103/2231-
4040.79796.
[22] Cheel, J.; Theoduloz, C.; Rodríguez, J.; Schmeda-
Hirschmann, G. Free Radical Scavengers and
Antioxidants from Lemongrass (Cymbopogon Citratus
(DC.) Stapf.). J. Agric. Food Chem. 2005, 53, 2511–2517.
DOI: 10.1021/jf0479766.
[23] Chen, Y.; Yu, H.; Wu, H.; Pan, Y.; Wang, K.; Jin, Y.;
Zhang, C. Characterization and Quantification by
LC-MS/MS of the Chemical Components of the
Heating Products of the Flavonoids Extract in Pollen
SEPARATION SCIENCE AND TECHNOLOGY 13
Typhae for Transformation Rule Exploration.
Molecules. 2015, 20, 18352–18366. DOI: 10.3390/
molecules201018352.
[24] Florindo, C.; Oliveira, F.; Rebelo, L.; Fernandes, A. M.;
Marrucho, I. Insights into the Synthesis and Properties
of Deep Eutectic Solvents Based on Cholinium Chloride
and Carboxylic Acids. ACS Sustain. Chem. Eng. 2014, 2,
2416–2425. DOI: 10.1021/sc500439w.
[25] Kujala, T. S.; Vienola, M. S.; Klika, K. D.;
Loponen, J. M.; Pihlaja, K. Betalain and Phenolic
Compositions of Four Beetroot (Beta Vulgaris)
Cultivars. Eur. Food Res. Technol. 2002, 214, 505–510.
DOI: 10.1007/s00217-001-0478-6.
[26] Marcus, Y. Extraction by Subcritical and Supercritical
Water, Methanol, Ethanol and Their Mixtures.
Separations. 2018, 5, 4. DOI: 10.3390/
separations5010004.
[27] Huang, Y.; Feng, F.; Jiang, J.; Qiao, Y.; Wu, T.;
Voglmeir, J.; Chen, Z.-G. Green and Efficient
Extraction of Rutin from Tartary Buckwheat Hull by
Using Natural Deep Eutectic Solvents. Food Chem.
2017, 221, 1400–1405. DOI: 10.1016/j.
foodchem.2016.11.013.
[28] Alberti, A.; Zielinski, A. A. F.; Zardo, D. M.;
Demiate, I. M.; Nogueira, A.; Mafra, L. I. Optimisation
of the Extraction of Phenolic Compounds from Apples
Using Response Surface Methodology. Food Chem.
2014, 149, 151–158. DOI: 10.1016/j.
foodchem.2013.10.086.
[29] Giusti, F.; Caprioli, G.; Ricciutelli, M.; Vittori, S.;
Sagratini, G. Determination of Fourteen Polyphenols
in Pulses by High Performance Liquid
Chromatography-diode Array Detection
(HPLC-DAD) and Correlation Study with Antioxidant
Activity and Colour. Food Chem. 2017, 221, 689–697.
DOI: 10.1016/j.foodchem.2016.11.118.
[30] Do, Q. D.; Angkawijaya, A. E.; Tran-Nguyen, P. L.;
Huynh, L. H.; Soetaredjo, F. E.; Ismadji, S.; Ju, Y.-H.
Effect of Extraction Solvent on Total Phenol Content,
Total Flavonoid Content, and Antioxidant Activity of
Limnophila Aromatica. J. Food Drug Anal. 2014, 22,
296–302. DOI: 10.1016/j.jfda.2013.11.001.
[31] Xu, Z.; Feng, S.; Shen, S.; Wang, H.; Yuan, M.; Liu, J.;
Huang, Y.; Ding, C. The Antioxidant Activities Effect of
Neutral and Acidic Polysaccharides from Epimedium
Acuminatum Franch. On Caenorhabditis Elegans.
Carbohydr. Polym. 2016, 144, 122–130. DOI: 10.1016/j.
carbpol.2016.02.041.
[32] Venditti, A.; Frezza, C.; Sciubba, F.; Serafini, M.;
Bianco, A.; Cianfaglione, K.; Lupidi, G.; Quassinti, L.;
Bramucci, M.; Maggi, F. Volatile Components, Polar
Constituents and Biological Activity of Tansy Daisy
(Tanacetum Macrophyllum (Waldst. Et Kit.) Schultz
Bip.). India CRO Prod. 2018, 118, 225–235. DOI:
10.1016/j.indcrop.2018.03.056.
[33] Mehmood, N.; Zubaır, M.; Rızwan, K.; Rasool, N.;
Shahid, M.; Ahmad, V. U. Antioxidant, Antimicrobial
and Phytochemical Analysis of Cichoriumintybus Seeds
Extract and Various Organic Fractions. Iran. J. Pharm.
Res. 2012, 11, 1145.
[34] Chandrasekaran, M.; Venkatesalu, V. Antibacterial and
Antifungal Activity of Syzygium Jambolanum Seeds.
14 Z. NASEEM ET AL.
J. Ethnopharmacol. 2004, 91, 105–108. DOI: 10.1016/j.
jep.2003.12.012.
[35] Thoo, Y. Y.; Ho, S. K.; Liang, J. Y.; Ho, C. W.; Tan, C. P.
Effects of Binary Solvent Extraction System, Extraction
Time and Extraction Temperature on Phenolic
Antioxidants and Antioxidant Capacity from
Mengkudu (Morinda Citrifolia). Food Chem. 2010,
120, 290–295. DOI: 10.1016/j.foodchem.2009.09.064.
[36] Ramadoss, G.; Muthukumar, K. Ultrasound Assisted
Metal Chloride Treatment of Sugarcane Bagasse for
Bioethanol Production. Renewable Energy. 2016, 99,
1092–1102. DOI: 10.1016/j.renene.2016.08.003.
[37] AlOmar, M. K.; Hayyan, M.; Alsaadi, M. A.; Akib, S.;
Hayyan, A.; Hashim, M. A. Glycerol-based Deep
Eutectic Solvents: Physical Properties. J. Mol. Liq.
2016, 215, 98–103. DOI: 10.1016/j.molliq.2015.11.032.
[38] Yin, X.; You, Q.; Jiang, Z.; Zhou, X. Optimization for
Ultrasonic-microwave Synergistic Extraction of
Polysaccharides from Cornus Officinalis and
Characterization of Polysaccharides. Int. J. Biol. Macromol.
2016, 83, 226–232. DOI: 10.1016/j.ijbiomac.2015.11.059.
[39] Unuigbe, C.; Enahoro, J.; Erharuyi, O.; Okeri, H.
Phytochemical Analysis and Antioxidant Evaluation of
Lemon Grass (Cymbopogon Citratus DC.) Stapf Leaves.
J. App. Sci. Environ. Manage. 2019, 23, 223–228.
[40] Jeganathan, P. M.; Venkatachalam, S.; Karichappan, T.;
Ramasamy, S. Model Development and Process
Optimization for Solvent Extraction of Polyphenols
from Red Grapes Using Box–Behnken Design. Prep.
Biochem. Biotechnol. 2014, 44, 56–67. DOI: 10.1080/
10826068.2013.791629.
[41] Belwal, T.; Dhyani, P.; Bhatt, I. D.; Rawal, R. S.;
Pande, V. Optimization Extraction Conditions for
Improving Phenolic Content and Antioxidant Activity
in Berberis Asiatica Fruits Using Response Surface
Methodology (RSM). Food Chem. 2016, 207, 115–124.
DOI: 10.1016/j.foodchem.2016.03.081.
[42] Bezerra, M. A.; Santelli, R. E.; Oliveira, E. P.; Villar, L. S.;
Escaleira, L. A. Response Surface Methodology (RSM)
as a Tool for Optimization in Analytical Chemistry.
Talanta. 2008, 76, 965–977. DOI: 10.1016/j.
talanta.2008.05.019.
[43] Gan, C.-Y.; Latiff, A. A. Optimisation of the Solvent
Extraction of Bioactive Compounds from Parkia
Speciosa Pod Using Response Surface Methodology.
Food Chem. 2011, 124, 1277–1283. DOI: 10.1016/j.
foodchem.2010.07.074.
[44] Aiyegoro, O. A.; Okoh, A. I. Preliminary Phytochemical
Screening and in Vitro Antioxidant Activities of the
Aqueous Extract of Helichrysum Longifolium DC.
BMC Complementary Altern. Med. 2010, 10, 21. DOI:
10.1186/1472-6882-10-21.
[45] Bubalo, M. C.; Ćurko, N.; Tomašević, M.; Ganić, K. K.;
Redovniković, I. R. Green Extraction of Grape Skin
Phenolics by Using Deep Eutectic Solvents. Food
Chem. 2016, 200, 159–166. DOI: 10.1016/j.
foodchem.2016.01.040.
[46] Curti, V.; Capelli, E.; Boschi, F.; Nabavi, S. F.;
Bongiorno, A. I.; Habtemariam, S.; Nabavi, S. M.;
Daglia, M. Modulation of Human miR-17–3p
Expression by Methyl 3-O-methyl Gallate as
Explanation of Its in Vivo Protective Activities. Mol.
Nutr. Food Res. 2014, 58, 1776–1784. DOI: 10.1002/
mnfr.201400007.
[47] Bednarek, P. Chemical Warfare or Modulators of
Defence Responses–the Function of Secondary
Metabolites in Plant Immunity. Curr. Opin. Plant
Biol. 2012, 15, 407–414. DOI: 10.1016/j.
pbi.2012.03.002.
[48] Bouaziz, A.; Mhalla, D.; Zouari, I.; Jlaiel, L.; Tounsi, S.;
Jarraya, R.; Trigui, M. Antibacterial and Antioxidant
Activities of Hammada Scoparia Extracts and Its
Major Purified Alkaloids. S. Afr. J. Bot. 2016, 105,
89–96. DOI: 10.1016/j.sajb.2016.03.012.