Separation Science and Technology

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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.

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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]

Nature has been a resource of remedial compounds for
The deep eutectic solvents (DESs) came into view as
thousands of years and huge number of advanced drugs
a new green substitute of traditional toxic organic sol-
have been extracted and isolated from green
vents and have been increasingly used in different
resources.[10] The phytochemicals of medicinal plants
research areas.[1] They composed of a mixture of hydro-
gen bond acceptor and donor species having supramo- have potential to combat against infectious and chronic
lecular structure with melting point significantly lesser diseases.[11,12] It is imperative to investigate the signifi-
than individual components.[2–4] These solvents have cance of widespread flora as they constitute traditional
similar physico-chemical properties of ionic liquids but and advanced therapeutic medicines. The extraction of
are accessible at low cost and biologically degradable.[5] medicinally active components from plants and their
The DESs can be prepared easily without using harsh evaluation (quantitative and qualitative) is imperative
organic solvents, which cause environmental pollution. for exploration of new potent drugs. The choice of
They have been attributed as environment-friendly sub- solvent, extraction technique and process parameters
stituents of conventional volatile organic solvents. (time, temperature, solid to liquid ratio, etc.) are most
Additionally, they are employed for the extraction and imperative factors for the separation of biologically
separation of bioactive ingredients (secondary metabo- active components from plants during extraction
lites) from medicinal plants.[3] Various DESs, which procedure.[13] The sonication method is considered
contain choline chloride with different hydrogen bond green extraction method due to short extraction time,
donors, were screened for the extraction of natural pro- less solvent consumption; reduced energy and signifi-
ducts and proved efficient as compared to the organic cant yield of extracted ingredients. The bubbling and
solvents for the extraction and separation of cavitation phenomena during ultrasonic process
anthocyanin[6] and phenolic compounds.[7,8] increase pressure and temperature which cause collapse
About 65–80% population of world relies on thera- of the cavities near the plant materials and ultimately
peutic foliage for their prime health concern, due to 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- Dr. Mansoor Hameed, Associate Professor of
icals into the solvent.[14,15] Department of Botany, University of Agriculture,
The optimization of process parameters is done using Faisalabad, Pakistan. The plant material was cleaned
empirical and statistical approaches. In classical study, with tap water to remove the dirt particles and dried in
the response is direct function of single variable but the shade (25 ± 5 °C) approximately for 30 days. The dried
actual response is produced due to interaction among material was ground using mechanical grinder, sieved to
diverse factors. On the other hand, statistical evaluation achieve the mesh size of 250 µm. The voucher sample
due to interactive influence is more reliable than classi- was kept in air tight container, assigned with number,
cal methods.[16] The response surface methodology and stored at ambient temperature (25 ± 5 °C) for
(RSM) facilitates to evaluate interactive effects of inde- further analysis.
pendent variables on the response variables. Therefore,
RSM is supportive for building models, designing
Chemicals and reagents
experiment, to find the linear and interactive effects of
several parameters simultaneously to obtain optimum Choline chloride (99%, Dae-Jung, Siheung, Korea), gly-
conditions.[17] cerol (99.50%, Dae-Jung, Siheung, Korea), Folin-
Cymbopogon citratus Stapf belongs to the family of Ciocalteu reagent (99.99%, Sigma-Aldrich, Darmstadt,
Poaceae and universally known as lemongrass which is Germany), DPPH (99.50%, Sigma-Aldrich, Darmstadt,
extensively used in customary medication.[18–20] The Germany), aluminum chloride (99.99%, Sigma-Aldrich,
reported ethnomedicinal compounds of C. citratus are Darmstadt, Germany), sodium carbonate (99%,
phenolics and flavonoids which consist of quercetin, api- Honeywell Riedel-deHaen, Seelze, Germany), potassium
ginin, luteolin, isoorientin 2ʹ-O-rhamnoside, and acetate (Sigma-Aldrich, 99%, Darmstadt, Germany),
kaempferol.[21] The C. citratus presents venerable remedy nutrient agar (99%, HiMedia, Pennsylvania, USA),
for headache, cough, flu, malaria, pneumonia, and for potato dextrose agar (99%, TM Media PR, London,
muscular disorder. The lemongrass is a good antiseptic UK), sabarose dextrose broth (99%, AppliChem
that smooths the process of detoxification of pancreas, GmbH, Darmstadt, Germany), resazurin (99% WRH
kidney, liver, gall bladder, and the digestive tract.[22] Int. Limited Harlow, UK).
The aim of present study was to investigate the effi- Analytical reference standards of rutin, primulaverin,
ciency of DES over aqueous methanol for the better sinapic acid, caffeic acid, quercetin, quercetin
extraction of phytochemicals. Choline chloride-glycerol- 3-O-glucuronide, chlorogenic acid, chrysoeriol 7-ruti-
based DES was prepared and used for the extraction of noside, apigenin-6-C-glucoside, calycosin with more
phenolics/flavonoids from C. citrates using maceration as than ≥98.00% purity were purchased from the Sigma
well as ultrasonic-assisted extraction (UAE). The initial Aldrich. Stock solutions (1.0 mg/mL) were prepared in
optimization of process parameters, i.e., time, tempera- methanol. Dilution of mixed standards was carried out
ture, and solid to liquid ratio was carried out by conven- with acetonitrile/water (18/82, v/v). Working standard
tional study followed by statistical evaluation of solutions mixture (1.0 µg/mL) for validation were pre-
phytochemicals (TPC, TFC, and DPPH) through Box- pared from stock solutions of particular analytical
Bhenken design (BBD) by RSM. In vitro antibacterial standard.[23]
and antifungal activities of plant extracts were assessed
by measuring inhibition zone and minimum inhibition
Synthesis of DES
concentration (MIC). Different bioactive components
have been characterized using LC-ESI-MS. The current The choline chloride and glycerol used for the prepara-
scientific study provides excellent alternative solvent for tion of DES, were oven dried at 60 °C for 24 h before use.
the extraction and separation of bioactive compounds These individual components, i.e., choline chloride and
with high yield from medicinal plants. 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]
Material and methods Hydrogen-bonded supramolecular structure of choline
chloride and glycerol is shown in Fig. 1.
Collection and preservation of plant material
Cymbopogon citratus Stapf plant as a whole was har-
Extraction of phytochemicals
vested in mid-October, 2016 from New Botanical
Garden (NBG) of University of Agriculture, Faisalabad, In the maceration process, 2 g dried powder of
Pakistan. The valid identification was conceded by 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

C. citratus was carried out using ChCl:Gly DES and
Antibacterial activity aqueous methanol. Maceration and UAE were applied
The antibacterial potential of C. citratus extracts against for the extraction purpose. The optimization of process
S. aureus (Gram-positive) and E. coli (Gram-negative) parameters (time, temperature, and solid to liquid ratio)
bacterial strains was investigated. The antibacterial was done. The yield of phytochemicals significantly
activity of plant extracts was evaluated by well diffusion depends on the extraction methods and solvent/media
assay. Minimum inhibitory concentration (MIC) for used.
each extract was determined by Broth micro-dilution
method.[33] Effect of time
The optimum extraction time in maceration and UAE
Antifungal activity would be very effective for the maximum extraction of
Two fungal strains F. solani and A. niger were used to phytochemicals. The preliminary study was performed
test the antifungal potential of C. citratus extracts. MIC for 3 to 24 h at 60 °C with 1 g/10 mL solid to liquid ratio
of plant extracts against fungal strains was determined during maceration. The highest extraction yield of
by following the already reported protocol with some 297 mg/g (in 9 h) and 193 mg/g (in 15 h) were observed
 SEPARATION SCIENCE AND TECHNOLOGY 5

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 ChCl:Gly and aq. MeOH with extraction yield of
change was observed up to 24 h as shown in Fig. 2(a). 250 mg/g and 145 mg/g, respectively, as shown in Fig.
The Fick’s second law of diffusion describes that the 2(e). With the increase in temperature, the diffusion of
solute in the matrix of plant equilibrate with the bulk phytochemicals increased while the viscosity of the sol-
of solvent around it. The change in time of solvent- vent decreased. As reported in literature, the viscosity of
solute equilibrium and concentration of phenolics ChCl:Gly is decreased from 281 cP to 79 cP for the rise
depends on the diffusion rate which is based on the in temperature from 298 K to 353 K.[37] Hence, the
solubility of phytochemicals in solvent.[35] In UAE, the solubility of phytochemicals increased with the rise in
extraction was carried out for 15–60 min and results are temperature during extraction. However, the decompo-
as shown in Fig. 2(d). The ChCl:Gly extract quantity sition of phenolics limits the increase in temperature. It
increased (229 mg/g) up to 60 min while the optimized was observed that moderate temperature provided posi-
time of aq. MeOH was 45 min with 126 mg/g yield. The tive effects on the extraction of phytochemicals yield.
cavitation effects increased with the increase of time in
UAE, which ultimately enhanced the mass transfer and Effect of solid to liquid ratio
rate of extraction.[36] The yield largely depends on the solid to liquid ratio so
its optimization is also necessary for the maximum
Effect of temperature extraction. The extraction by maceration and UAE was
The temperature is also a significant factor to assist the done using 0.5–2 g/10 mL solid to liquid ratio with
extraction process. In maceration, the extraction was respective optimized conditions of time and tempera-
carried out at 30–70°C. It was evaluated that the yield ture. In maceration, as shown in Fig. 2(c) the optimum
was increased with the rise in temperature up to 60°C yields of 299 mg/g was recorded with ChCl:Gly solvent
(294 mg/g) and then remained constant (295 mg/g) at 70 at 1.0 g/10 mL and then continuous decrease was
°C. In case of aq. MeOH, the optimized temperature 60 ° observe up to 2.0 g/10 mL while the aq. MeOH showed
C with 230 mg/g yield was observed as shown in Fig. 2 220 mg/g yield with 1.5 g/10 mL solid to liquid ratio then
(b). In UAE, the temperature effect was studied at 40–70 became constant. In UAE, both the ChCl:Gly and the aq.
°C and obtained temperature was 60 °C for the both 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 and antioxidant activity. In UAE, the interactions
Fig. 2(f). The rate of diffusion of solute molecules into between independent variables are illustrated in contour
solvent was directly influenced by the solid to liquid surfaces as shown in Fig. 4. The upper and lower levels
ratio. A small solid to liquid ratio can dissolve bioactive of time, temperature, and solid to liquid ratio did not
ingredients with more efficacy, leading to an enhance show the valuable results. The medium levels of process
extraction yield.[38] variables preferential for effective extraction.
It has been observed that the extraction efficacy is The inconsistency between the predicted and the
primarily based on the dissolving ability of solvents. experimental values is identified as residual and it
Phenolic compounds are generally polar and solvent imparts momentous role to judge the significance of
appears to play a significant role in their extraction so applied model. Therefore, if the statistical model is well
that polar solvents tend to contain more of these com- fitted, its residuals graphs present a behavior that por-
ponents compared to the less polar or nonpolar trays a normal distribution.[42] The graphs of normal %
solvents.[39] The DES have strong hydrogen bonding, probability versus studentized residuals of all three pro-
which ultimately improved the extraction efficiency by cess parameters of both extraction methods are pre-
increasing the dissolution level.[3] The prepared DES sented in Figure S2-S4 (supplementary information).
showed enhanced extraction yield compared with aqu- The perturbation plots demonstrate the deviation of
eous methanol. 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
Response surface methodology
(B) solid to liquid ratio (C) act as the controlling factors
The optimization of extraction time, temperature, and for the utmost yield. The perturbation plots are given in
solid to liquid ratio (independent variables) was exam- supplementary information Figure S5-S7.
ined comprehensively using BBD.[40] Three responses, The extraction through maceration process was
TPC, TFC, and % inhibition of DPPH were evaluated by investigated at different conditions of time (6–12 h),
applying the second-order polynomial equation. The temperature (40–80 °C) and solid to liquid ratio
fairly high regression coefficient (R2) values for TPC, (0.5–1.5 g/10 mL), which were chosen on the basis
TFC, and DPPH with least standard deviations during of preliminary experiments. It was found that the
maceration as well as UAE described applied regression highest values of TPC (95 ± 2.75 mg GAE/g), TFC
model was well fitted. The validation of quadratic model (86 ± 3.0 mg QE/g), and DPPH (89%±2.00) were
with small C.V (less than 5% in all responses) explained achieved at 60 °C after 9 h of maceration with 1 g/
the better reliability and good precision of data. The lack 10 mL solid to liquid ratio. The experimental values
of fit with more than 0.05 p-values also illustrated the were in good agreement with those of predicted
validity of applied quadratic model in both extraction (shown in Table 2), thus indicating adequacy of the
methods.[41] Sum of squares (SS) relate to the total developed model. It is considered that high tempera-
variance of the observations. The sample variance is ture leads to decomposition of phenolic contents and
also referred to as a mean square (MS) because it is more extraction time leads to the unwanted oxidation
obtained by dividing the sum of squares by the respec- of these compounds. The higher solid to liquid ratio
tive degrees of freedom (df). In Tables 3 and 5, the SS, reduced the quantitative yield because less amount of
MS, and df values of analysis of variance are given. The solvent is available for solubilization.[43] Consequently,
interaction between independent variable and their the moderate conditions are necessary for maximum
mutual influence on the response values is depicted by and safe recovery of these compounds. The ANOVA
3D response surfaces. The contours represent the inter- of these experiments, following quadratic model equa-
actions between independent variables and responses, tions in term of coded variables, showed the effect of
keeping other independent variable at fixed level. The operating parameters for all three responses is given in
independent variables, i.e., time (A), temperature (B) Table 3.
and solid to liquid ratio (C) with their combined influ- For the UAE, the extraction was conducted at three
ences (AB, BC, and AC) on the extraction of phenolic different levels for each of time (30–60 min), tempera-
and flavonoids contents and % inhibition of DPPH ture (50–70 °C), and solid to liquid ratio (1.0–2.0 g/
during maceration were depicted in response surfaces 10 mL). The highest values of TPC (135 ± 2.50 mg
as shown in Fig. 3. It is observed that time, temperature, GAE/g), TFC (93 ± 2.50 mg QE/g), and DPPH
solid to liquid ratio and their interactions have positive (94 ± 1.75%) were observed in 45 min under 60 °C
effects upto some extent and then decline started. The with 1.5 g/10 mL solid to liquid ratio. The experimental
medium points showed maximum yield of TPC, TFC, values of TPC, TFC, and DPPH showed good agreement
 SEPARATION SCIENCE AND TECHNOLOGY 7

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 and quadratic model equations in terms of coded vari-
DPPH activity was maximum when the TPC and TFC ables for all three responses are given Table 5. It is
contents were the highest in extracts. This shows posi- concluded that model linear terms (A, B, C), interaction
tive correlation between TPC, TFC, and DPPH activity. terms (AB, AC, BC), and quadratic terms (A2, B2, C2) are
The ability of plant extract to scavenge DPPH also influencing and show level of significance with p value
reflects its potential to inhibit the formation of free <.05 for all response variables.
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 Antimicrobial activities
well as sonication process played a major role to extract Phytochemicals constituents have precious curative
the higher content of phenolics and flavonoids.[45] It has agents to treat the human being diseases with supple-
been observed from the results of RSM treatment that mentary efficacies having minimum or no side
TPC TFC and DPPH showed utmost quantitative yield effects.[46] A massive amount of scientific study proved
and activity at the same conditions which has shown that plants bioactive constituents have aptitude to pro-
maximum crude yield (in preliminary studies). The duce antimicrobial agents as defense mechanism to
ANOVA (for experiments conducted through UAE) 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 for all extracts along with standard Terbinafine are
check the potential of extracts against these microorgan- shown in Table 6.
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 Identification of bioactive compounds through
evaluated. The antibacterial activities were assessed quan- LC-ESI-MS
titatively by measuring the inhibition zones. The MIC by The mass spectral identification of phenolics and flavo-
micro-dilution method of all extracts were also deter- noids structures is widely carried out with electron spray
mined, which showed the inhibition zone.[48] The ChCl: ionization (ESI) technique. This ionization technique has
Gly extracts have shown more potential to extinct both been applied to identify phenolics flavonoids. Complex
Gram-positive (S. aureus) and Gram-negative (E. coli) bac- fragmentation may occur with ESI due to the broad
terium than aq. MeOH extracts in maceration and UAE. spread of internal energy containing the initially pro-
The ChCl:Gly extract obtained by UAE showed larger zone duced M+• ions, which may hide or suppress the M+•
of inhibition (40.5 ± 1.5 mm and 39.5 ± 1.4 mm) with ions and significant primary fragment ions. The chemical
minimum MIC (12.5 ± 1.5 µg/mL and 25 ± 2.0 µg/mL) structures of identified compounds are given in Figure S8.
against S. aureus and E. coli, respectively. The plant extracts The identified compounds in this study were Primiverin/
showed the antibacterial potential but less than that of Primulaverin, sinapic acid, rutin, quercetin
standard Rifampicin. The results are shown in Table 6. 3-O-glucuronide, chrysoeriol 7-rutinoside, apigenin-
6-C-glucoside, and calycosin. The Primiverin/
Antifungal activity Primulaverin was identified with Rt 8.1 min and detected
Two fungal strains, i.e., F. solani and A. niger were used as [M + H]+ ion at m/z 475.2. The sinapic acid with Rt
to assess the potential of C. citratus against these fungal 8.15 min was identified as [M + H]+ ion at m/z 224.1. The
agents. The antifungal activity of extracts obtained rutin with Rt 24.57 min was identified as [M + H]+ ion
through maceration and UAE (at optimized conditions at m/z 611.4. Quercetin 3-O-glucuronide was identified as
in both cases) was also quantitatively determined by [M + H]+ ion at m/z 478.1 with Rt of 20.28 min and
measuring the inhibition zones as well as the MIC. The Chrysoeriol 7-rutinoside was identified as [M + H]− ion
ChCl:Gly extracts proved more potential to destroy at m/z 608.1 with 45.324 min. Apigenin-6-C-glucoside
F. solani and A. niger fungi as compared to aq. MeOH with Rt of 18.11 min was identified at m/z 342.2.
extracts in both maceration and UAE. In comparison Calycosin with Rt of 3.43 min was identified as
with aq. MeOH extracts, the ChCl:Gly extracts obtained [M + H]+ ion at 285.1. The LC-ESI-MS spectra of
by applying UAE showed larger inhibition zone C. citratus are shown in supplementary Figures S9–S15.
(41 ± 1.4 mm and 34 ± 1.4 mm) with MIC The phenolics and flavonoids extracted from C. citratus
(12.5 ± 1.0 µg/mL and 12.5 ± 1.5 µg/mL) against 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 Funding
It is concluded from this study that choline chloride- This work was supported by the Higher Education
glycerol binary solvent proved better alternative for the Commission, Pakistan [20-2(8)/ASIP/R&D/HEC/17/00038.].
extraction of phytochemicals from C. citratus compared
with aqueous methanol. RSM with three factors at three References
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