South African Journal of Botany 148 (2022) 238
250

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

In-vitro antimicrobial, antioxidant, anti-inflammatory, and cytotoxic

activities of Populus ciliata bark and leaves: A comparative study
Ishita Guleriaa, Amita Kumaria,*, Marie-Aleth Lacaille-Duboisb,*, Adesh K. Sainic, Vikas Kumard,
Reena V. Sainic, Uma Ranjan Lale, Naseem A. Gaurf, Sonam Kumarif, Amit Sethg,
Jyoti Dhatwaliaa, Shabnam Thakura, Sohan Lala
a
School of Biological and Environmental Sciences, Faculty of Sciences, Shoolini University, Solan, Himachal Pradesh 173229, India
b
Department of Pharmacognosy, CSGA (Agrosup/CNRS/INRAE/UBFC), UFR of Health Sciences, University of Bourgogne Franche-Comte
, Dijon, France
c
Department of Biotechnology, MMEC, Central Research Cell, Maharishi Markandeshwar (Deemed to be University), Mullana, Haryana 133203, India
d
University Institute of Biotechnology, Chandigarh University, Gharuan, Mohali, Punjab 140413, India
e
Department of Natural Products, NIPER-A, Opposite Air Force Station, Palaj, Gandhinagar, Gujarat 382355 India
f
Yeast Biology Group, DBT-ICGEB Centre for Advanced Bio Energy Research, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,
New Delhi 110067, India
g
Department of Life Sciences (Botany), Manipur University, Imphal 795003, India

A R T I C L E I N F O A B S T R A C T

Article History: Populus ciliata Wall. ex Royle (Salicaceae family) is a fast-growing, multipurpose native tree of the Himalayas.
Received 11 November 2021 Traditionally, the tree's bark is used for blood purification, treatment of rheumatism, and fatigue, and to
Revised 20 April 2022 relieve the pain of menstrual cramps. The present study deals with screening crude methanolic extracts and
Accepted 21 April 2022
fractions (n-hexane, chloroform, ethyl acetate, and n-butanol) of P. ciliata bark and leaves for total polyphe-
Available online 4 May 2022
nolic contents, antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activities. Results showed that
Edited by Dr. S.V. Vuuren among all tested samples, the bark ethyl acetate fraction had the highest polyphenolic contents and strong
antioxidant potential (DPPH 25.75§3.60 mg/mL; ABTS 33.55§2.96 mg/mL; FRAP 70.39§1.75 mM of Fe equiv-
Keywords:
alents). The HPTLC analysis confirmed the presence of gallic acid, salicin, and quercetin molecules in the
Populus ciliata
extracts and different fractions of P. ciliata whereas ethyl acetate fractions showed their maximum quantity.
Biological activities
The ethyl acetate fraction of bark also revealed higher antibacterial activity against Pseudomonas aeruginosa
HPTLC
Polyphenolic content (MIC 0.0156 mg/mL), Klebsiella pneumoniae and Staphylococcus aureus (MIC 0.0312 mg/mL), Bacillus subtilis
Ethnomedicinal use and Escherichia coli (MIC 0.0625 mg/mL), and antifungal activity against Fusarium oxysporum (MIC 0.125 mg/
mL) and Rosellinia necatrix (0.250 mg/mL), whereas butanol fraction of bark showed maximum antifungal
activity against Candida auris (MIC 1.25 mg/mL). The highest anti-inflammatory activity was observed in
bark ethyl acetate fraction (IC50 0.113§0.017 mg/mL for egg albumin and 0.160§0.014 mg/mL for bovine
serum albumin assay) in comparison with other extracts and fractions. Additionally, bark crude extract and
its ethyl acetate fraction were observed more effective against colon cancer cell lines (IC50 values <0.170 mg/
mL for HCT-116 and <0.340 mg/mL for SW-620). The study concludes that the bark ethyl acetate fraction is
the most biologically active fraction due to a higher number of polyphenolic contents (salicin, gallic acid, and
quercetin) and among both plant parts, bark has a higher medicinal value than leaves. Therefore, this fraction
can be further explored to extract biologically active compounds of pharmaceutical, food, and cosmetic val-
ues.
© 2022 SAAB. Published by Elsevier B.V. All rights reserved.

Abbreviations: (NH4)2S2O₈, Ammonium per sulfate; C, Degree Celsius; mg, Microgram; mL, Microliter; mM, Micro molar; ABTS, 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid; AlCl3, Aluminium chloride; CFU, Colony forming unit; CH3COONa, Sodium acetate; cm, Centimetre; CO-2, Carbon dioxide; ddH2O, Double distilled water; dH2O, Distilled water;
DMSO, Dimethyl sulphoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, Dry weight; etc., et cetera; FeCl3.6H2O, Ferric chloride hexahydrate; FBS, Fetal Bovine Serum; FeCl3.6H2O,
Ferric chloride; FeSO4, Ferrous sulfate; FRAP, Ferric reducing antioxidant power; g, Gram; GAE, Gallic acid equivalents; IC50, Half-maximal inhibitory concentration; kg, Kilogram;
mg, Milligram; MIC, Minimum inhibitory concentration; min, Minutes; mL, millilitre; mm, millimetre; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NA,
Nutrient agar; nm, Nanometre; Na2CO3, Sodium carbonate; NaNO₂, Sodium nitrite; NaOH, Sodium hydroxide; NB, Nutrient broth; OD, Optical density; PDA, Potato dextrose agar;
pH, Potential of hydrogen; QUE, Quercetin trihydrate equivalents; r2, Coefficient of determination; rpm, Revolutions per minute; TFC, Total flavonoids content; TPC, Total phenolics
content; TPTZ, 2,4,6-Tri(2-pyridyl)-s-triazine; UV, Ultra-violet; w/v, Weight in volume; YPD, Yeast extract peptone dextrose; ZOI, Zone of inhibition
* Corresponding authors.
E-mail addresses: amitabot@gmail.com (A. Kumari), Marie-Aleth.Lacaille-Dubois@u-bourgogne.fr (M.-A. Lacaille-Dubois), sohanlal48510@gmail.com (S. Lal).

https://doi.org/10.1016/j.sajb.2022.04.040
0254-6299/© 2022 SAAB. Published by Elsevier B.V. All rights reserved.
I. Guleria, A. Kumari, M.-A. Lacaille-Dubois et al. South African Journal of Botany 148 (2022) 238
250

1. Introduction 2. Materials and methods

All plants or plant parts have medicinal value due to their 2.1. Plant identification and chemicals
characteristic chemical profile. Some plants or plant parts are
reported in the literature for their traditional medicinal use, The P. ciliata trees (34 years old; 82§2 cm stem girth diameter) in
whereas some are still waiting for their exploration. Hence, the field/sampling site (Sankant Mochan Mandir, district Shimla, H.P.,
screening unexplored plant or plant parts for their phytoconstitu- India) were selected for sample collection based on morphological
ents or their biological potential is gaining major attention day characters (gray color on the lower side of leaves and dark brown
by day around the world (Singh et al., 2019). On the other hand, color of bark with vertical cracks). The plant twigs were collected in
synthetic drugs that people are using to cure diseases have seri- July 2018 and used for herbarium preparation. The authentication of
ous side effects (Zhang et al., 2015; Srivastava et al., 2019). There- the plant was done in the Botanical Survey of India, Dehradun, with
fore, their use is decreasing nowadays, and people’s interest is accession number Acc-116. The authenticated sheets were also
shifting to herbal drugs. According to Chaudhary et al. (2020), the deposited in the herbarium of Shoolini University, Solan, with
demand for plant-based therapeutics and their products are voucher no. SUBMS\BOT-3885.
increasing annually to 15%
25% and WHO estimated to increase All the growth media, reagents, chemicals, and solvents were
that demand to 5 trillion USD in 2050. from Himedia Laboratories Pvt. Ltd., Mumbai.
In the literature, different plant species have been screened for
their chemical and pharmacological profiles to find alternative 2.2. Sample collection and extract preparation from the bark and leaves
sources of synthetic drugs (Katiyar et al., 2012). Most plant spe-
cies are rich in polyphenolic contents like phenolic acids, flavo- The bark and leaves (4 kg) of selected P. ciliata trees (82§2 cm
noids, phenolic glycosides, tannins, etc. These are secondary stem girth diameter) were collected in polyethylene bags and
metabolites synthesized during the natural physiological pro- brought to the laboratory for further analysis. The collected bark and
cesses (Kabera et al., 2014) and are known to reduce the cell's leaves were cut into small pieces and dried in the dark for 20 days at
oxidative stress and reported for anti-inflammatory, anticancer, room temperature. The dried plant parts were crushed using a mixer
and antimicrobial activities (Swallah et al., 2020; Efenberger- grinder (Philips HL7707/00) and stored in airtight glass bottles.
Szmechtyk et al., 2021). The genus Populus members (family Sali- The coarse powder of bark and leaves (50 g) were extracted sepa-
caceae) are also known to contain high amounts of polyphenols rately five times with methanol (500 mL) using a Soxhlet at 40 °C for
like phenolic acids, tannins, flavonoids, stilbenes, coumarins, cur- 72 h (Ghosh and Chandra, 2006). The filtration of extract was done
cuminoids, lignans, etc. (Devappa et al., 2015a; 2015b; through Whatman filter paper No. 41 and filtrate was further concen-
Tawfeek et al., 2019; Guleria et al., 2021). Among all the species trated at 37 °C using a Hot air oven. The 5 g of concentrated crude
of the genus Populus, P. ciliata Wall. ex Royle (Himalayan Poplar), extract of bark, termed here BCE was dissolved separately in 100 mL
is a multipurpose native tree of the Himalayas. Ethnomedicinally, of double-distilled water (ddH2O) for 15 min by stirring. The mixture
only the bark of the tree is reported to purify the blood, relieve was then transferred into the separating funnel and successively par-
menstrual cramps pain, and treat fatigue rheumatism titioned against an equal amount of hexane followed by chloroform,
(Kunwar and Adhikari, 2005; Masoodi et al., 2014), whereas ethyl acetate, and n-butanol solvents (Mehmood et al., 2012;
leaves are only used as fodder by the local people. Different phe- Kumar et al., 2018). The butanol fraction (BBF), ethyl acetate fraction
nolic compounds such as salicin, caffeic acid, populin, ferulic acid, (BEF), chloroform fraction (BCF), and hexane fraction (BHF) of bark
4-coumaric acid, etc. have been reported from the tree were collected separately, dried, and stored at 4 °C for further use.
(Greenaway and Whatley, 1991; Kumari et al., 2016). Through The same procedure was repeated with the crude extract of leaves
the GC
MS technique, Kumari et al. (2020) identified ascorbic (LCE) for fractionation and each fraction of leaves (LBF, LEF, LCF, LHF)
acid, 9,12-octadecadienoic acid, hexadecenoic acid, ethyl ester as was separately collected, dried, and stored at 4 °C for further use. For
major phytoconstituents in the methanolic extract of P. ciliata biological assays, each extract and fraction was dissolved in 10%
leaves which have been reported in the literature for antioxidant, DMSO (20 mg/mL, w/v).
antimicrobial, and anticancer properties (Manilal et al., 2009; The yield percentage of the bark and leaves’s crude extract and its
Fernandes et al., 2015; Abountiolas and do Nascimento fractions was calculated from the following equation given by
Nunes, 2018). Recently through HPLC, Saqib et al. (2021) reported Pavithra and Vadivukkarasi (2015):
the confirmation of p-coumaric acid, vanillic acid, catechin, and
Weight of dry extract
quercetin in the crude methanolic extract of leaves and stem of P. Yield ð%Þ ¼ X 100
Weight of sample used in extraction
ciliata.
Moreover, phenolic glycosides have already been proven with
biological properties like antimicrobial, antioxidant, anticancer, anti- 2.3. Phytoconstituent analysis
inflammatory, etc. (Zhang et al., 2006; Devappa et al., 2015a; 2015b;
Tawfeek et al., 2019). None of the reports is available in the literature 2.3.1. Total phenolic content (TPC)
where the bark of P. ciliata was used to screen for antioxidant, antimi- The quantification of TPC in BCE was performed by following the
crobial, anti-inflammatory, and anticancer activities. Only the leaves methodology of Singleton et al. (1999). In this method, BCE (0.1 mL)
methanolic extract of P. ciliata has been reported for antibacterial and its subsequent fractions (1 mg/mL in methanol, w/v) were mixed
activity (Kumari et al., 2020). Therefore, being rich in phenolic glyco- with Folin-Ciocalteu’s phenol reagent (2 ml; 1:1 diluted with dH2O)
sides and other phenolic compounds, the present study identifies and allowed to react for 10 min. After incubation, 7.5% Na2CO3 (1 mL)
active biological fractions from the crude methanolic extract of bark was added to the solution and the final volume was made to 3 mL by
and leaves of P. ciliata. As per the latest knowledge, this is the adding dH2O. The whole solution was then kept for 30 min in the
first study on the bark extracts of P. ciliata. The outcome of the dark, and OD at 760 nm was measured. TPC content was calculated
present study would be helpful in the identification of new bioac- by plotting the standard curve of gallic acid (0.020
0.100 mg/mL)
tive compounds (antioxidant and antimicrobial compounds) from with linear regression line (y = 0.0104x + 0.0075; r2 = 0.9884) and
the identified active fraction, which might be further explored in expressed in mg/g gallic acid equivalents (GAE). A similar method
the development of an effective drug system for the cure of sev- was repeated separately with triplicate BCE-fractions, LCE, and LCE-
eral diseases. fractions.
239
I. Guleria, A. Kumari, M.-A. Lacaille-Dubois et al. South African Journal of Botany 148 (2022) 238
250

The TPC in all extracts and fractions was calculated separately where Abs (C) was the absorbance of the control solution and Abs (S)
using the following equation: was the absorbance of the test solution.
For the calculation of IC50 value, test sample’s linear regression of
Conc: x Vol:
A¼ plots of different concentrations was used against the mean percent-
W
age of the antioxidant activity obtained from triplicate assays.
In this equation, A- TPC in mg/g GAE; Conc.- concentration of GAE
calculated from the calibration curve in mg/mL; Vol.- volume of
2.4.2. The radical scavenging assay using 2,20 -azino-bis (3-
extract in mL, and W- weight of extract in g.
ethylbenzothiazoline-6-sulfonic acid) (ABTS)
The ABTS assay was used to determine the radical scavenging
2.3.2. Total flavonoids content (TFC) activity of BCE by following the methodology of Maurya and Devasa-
The TFC of BCE was carried out through the aluminum chloride gayam (2010). The BCE at different concentrations (20
100 mg/mL)
method of Zhishen et al. (1999). To 0.1 mL of BCE (1 mg/mL in metha- was used separately to detect the radical percentage inhibition. In
nol), 5% NaNO₂ (0.3 mL) was added. After incubation of 5 min, 10% the first step, 7 mM of ABTS and 2.45 mM of (NH4)2S2O₈ (1:1) were
AlCl3 (0.3 mL) and 1 M NaOH (2 mL) were added. The solution was mixed and incubated in the dark for 16 h at room temperature. Then,
diluted to 10 mL by adding dH2O and was kept in the dark for the ABTS solution was diluted with methanol to the 0.700 absor-
15 min. After incubation, the optical density of the yellow color was bance. In the third step, 100 mL of each concentration was mixed
taken at 519 nm. TFC content was calculated from the standard curve with 900 mL of diluted ABTS solution into the test tubes. The test
of quercetin (QUE) (0.020
0.100 mg/mL) with linear regression tubes were incubated in the dark for 30 min and optical density was
graph line (y = 0.0106x + 0.0149; r2 = 0.9853) and expressed in mg/g taken at 734 nm. The results were recorded in triplicate and sepa-
QUE. A similar procedure was repeated separately with triplicate rately done with standard ascorbic acid (20
100 mg/mL), BCE-frac-
BCE-fractions, LCE, and LCE-fractions. tions, LCE, and LCE-fractions.
The TFC in all extracts was calculated separately using the follow-
ing equation: ½Abs ðCÞ
Abs ðSÞ
Scavenging activity % ¼ X 100
Abs ðCÞ
Conc: x Vol:
A¼
W where, Abs (C) was the absorbance of the control solution and Abs (S)
was the absorbance of the test solution.
In this equation, A- TFC in mg/g QUE; Conc.- concentration of QUE
For the calculation of IC50 value, test sample’s linear regression of
calculated from the calibration curve in mg/mL; Vol.- volume of
plots of different concentrations was used against the mean percent-
extract in mL, and W- weight of extract in g.
age of the antioxidant activity obtained from triplicate assays.

2.3.3. Quantification of gallic acid, salicin, and quercetin through hptlc

CAMAG HPTLC system consisting of an automatic Linomat V sam- 2.4.3. Ferric-reducing/antioxidant power (FRAP) radical scavenging
ple applicator, a CAMAG TLC scanner was used for simultaneous assay
quantitative estimation of phenolic compounds such as gallic acid, The FRAP radical scavenging assay of BCE was determined by fol-
salicin, and quercetin in all extracts and fractions of P. ciliata lowing the protocol of Benzie and Strain (1996). The 100 mL of differ-
(Alam et al., 2013; Chauhan et al., 2018). In this method, aluminum ent concentrations of BCE and its different solvent fractions
TLC plates (20 £ 20 cm, 0.20 mm thickness, with silica gel 60 with (20
100 mg/mL) were mixed with freshly prepared FRAP reagent
fluorescent indicator, Loba Chemie) were used as the stationary (900 mL) into the test tubes. The FRAP reagent was prepared by mix-
phase, and TLC plate visualization was done under UV chamber at ing CH3COONa buffer (300 mM; pH 3.6), TPTZ solution (10.0 mM),
254 nm. For the interpretation of results CATS 4 Software was used and FeCl3¢6H2O solution (20.0 mM) in a ratio of 10:1:1 in volume.
through densitometric evaluation of chromatograms. In addition to The test tubes were incubated for 30 min in the dark and optical den-
this, a stock solution [mixture of gallic acid, salicin, and quercetin sity was recorded at 593 nm. The results were recorded in triplicate.
(1:1:1)] was prepared in the methanol. After this, the standard spot- This method was repeated separately with ferrous sulfate
ting (200
1500 ng/spot) was done on the TLC plate. The 3 mL (20
100 mg/mL), standard ascorbic acid (20
100 mg/mL), BCE-frac-
(10 mg/mL) of each plant sample (BCE, BCE-fractions, LCE, and LCE- tions, LCE, and LCE-fractions in triplicate. The antioxidant capacity of
fractions) was separately applied on TLC plate and run in the solvent the sample was calculated from the linear calibration curve of ferrous
system containing toluene: ethyl acetate: methanol: formic acid sulfate (y = 0.56x-0.0045; r2 = 0.9589) and expressed as mM FeSO4
(4:3:2:0.5, v/v) at room temperature (25§2 °C). equivalents /gram of sample (DW). For the calculation of IC50 value,
the test sample’s linear regression of plots of different concentrations
was used against the mean percentage of the antioxidant activity
2.4. In-vitro antioxidant assays
obtained from triplicate assays.

2.4.1. Free radical scavenging assay using 2,2-diphenyl-1-

picrylhydrazyl (DPPH) 2.5. Antimicrobial assays
The DPPH assay of Mensor et al. (2001) was used to determine
BCE's free radical scavenging activity in different concentrations 2.5.1. Microbial organisms
(20
100 mg/mL). Firstly, the 100 mL of each concentration was sepa- Five standard bacterial type strains [two Gram's positive, viz.,
rately mixed with 900 mL of DPPH (0.004% in methanol, w/v) into the Staphylococcus aureus MTCC 737, and Bacillus subtilis MTCC 441 and
test tubes. The test tubes for 30 min were incubated in the dark and three Gram's negative viz., Klebsiella pneumoniae MTCC 109, Escheri-
optical density was measured at 517 nm using a UV
visible spectro- chia coli MTCC 739, and Pseudomonas aeruginosa MTCC 424] were
photometer. The procedure was repeated separately with standard used and purchased from Institute of Microbial Technology (IMTech),
ascorbic acid (20
100 mg/mL), BCE-fractions, LCE, and LCE-fractions Chandigarh, India. The bacteria were maintained on Nutrient Agar
in triplicate. (NA) at 37 °C for 18 to 24 h.
The radical scavenging activity was calculated as follows: The antifungal activity against three human pathogenic fungal
type strains [Candida albicans (SC 5314 ATCC MYA2876), C. glabarata
½Abs ðCÞ
Abs ðSÞ
Scavenging activity % ¼ X 100 (CBS 138 ATCC 2001), and C. auris (CBS 10913T)] and non-pathogenic
Abs ðCÞ
fungal type strain [Saccharomyces cerevisiae (BY4741, ATCC
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I. Guleria, A. Kumari, M.-A. Lacaille-Dubois et al. South African Journal of Botany 148 (2022) 238
250

4,040,002)] was done at DBT-ICGEB center for Advanced BioEnergy CE (20 mg/mL in 10% DMSO) was mixed with NB/PDB in a 96-well
Research, New Delhi, India. microtiter plate. Serial dilution of extracts was made so that the next
Additionally, antifungal activity against Rosellinia necatrix well had half the concentration from the previous well. The 10 mL of
HG964402.1 and Fusarium oxysporum SR266
9 were also observed. each bacterial/fungal strain solution (OD»0.1) was added separately to
The fungal strain was obtained from the Faculty of Biotechnology and the well of a 96-well micro titre plate then incubated at 37 °C for 24 h
Applied Sciences, Shoolini University, Solan, and culture was main- and 25§2 °C for 48 h for bacteria and fungi, respectively in an incuba-
tained on Potato Dextrose Agar (PDA) media at 25 °C. tor. After incubation, 15 mL of resazurin (0.04% in ddH2O) solution was
added to wells as an indicator. The color change in the well was
2.5.2. Antibacterial activity observed visually. This method was repeated separately with BCE-
The disk diffusion assay of Bauer (1966) was used to determine fractions, LCE, and LCE-fractions in triplicate.
the antibacterial activity of P. ciliata BCE against selected bacterial For the determinations of MIC of yeast strains, Wayne (2002)
strains. The 100 mL of each bacterial strain (OD»0.1) was spread uni- methodology was followed. The BCE was mixed with yeast extract
formly on the NA plate. In the next step, from stock solutions (20 mg/ peptone dextrose (YPD) broth separately in a 96-well microtiter
mL in 10% DMSO), BCE in different concentrations (0.4
1.0 mg/mL plate. Serial dilutions of extracts were made so that the next well had
per disk) was separately poured on a 4 mm discs in the nutrient agar half the concentration from the previous well. Fungal strains were
plate. The plate was then placed in the refrigerator for 2 h. In the last then inoculated with inoculum size 1 £ 104 CFU/mL. Cells were
step, the plate at 37 °C was incubated for 24 h in the incubator. Ampi- allowed to grow for 48 h, then OD at 600 nm was recorded by UV-vis-
cillin (0.100 mg/mL) was used as a positive control against all tested ible Spectrophotometer and MIC was determined by comparing rela-
strains and DMSO (10%) was used as a negative control. The zone of tive growth without extract condition. The same procedure was
inhibition (ZOI) was measured in mm with an antibiotic zone scale. repeated separately with BBF, BEF, LCE, LBF, LEF in triplicate. A strain
The results were obtained in triplicate. This method was repeated could either be susceptible, non-susceptible, or resistant against the
separately for BCE fractions, LCE, and LCE fractions in triplicate. tested extracts.

2.5.3. Antifungal activity 2.6. In-vitro anti-inflammatory analysis

2.5.3.1. Poison food technique (PFT). The PFT was used to determine 2.6.1. Bovine albumin protein denaturation assay
the antifungal activity of P. ciliata bark extract and various fractions The BCE at different concentrations (0.2
1 mg/mL) were used to
by following the protocol of Grover and Moore (1962). Each fungal analyze the anti-inflammatory activity per Gunathilake et al. (2018).
strain was placed on PDA media Petri plates containing BCE extract To 200 mL of BSA (1%), 2.8 mL of PBS (pH 6.4) was added. Then 2 mL
in different concentrations (0.4
1.0 mg/mL per plate from the of each concentration of BCE and its fractions (0.2
1 mg/mL) were
20 mg/mL in 10% DMSO stock solution) and incubated at 25 °C for separately added to it. The reaction mixture was further allowed to
7 days. The colony diameter was measured through an antibiotic react at 37§2 °C in an incubator for 15 min. After that, the mixtures
zone scale. Media without the extract was used as negative control were heated at 70 °C for 5 min in a hot water bath. The absorbance of
and hygromycin-b (0.100 mg/mL in dH2O) was used as a positive the reaction solution was measured at 660 nm against blank after
control. A similar method was repeated with BCE fractions, LCE and cooling. Diclofenac sodium (0.2
1 mg/mL) was used as reference
LCE-fractions in triplicate. The efficacy of each plant extract for anti- drug. The experiment was replicated thrice. The same procedure was
fungal effect was expressed in terms of percentage inhibition and cal- repeated separately with BCE-fractions, LCE, and LCE-fractions in
culated by using the following formula: triplicate. The IC50 value was calculated from the linear regression of
Colony diameter of control
Colony diameter of extract plots of different concentrations of the test sample against the mean
% Inhibition ¼ X 100 percentage inhibition of the anti-inflammatory activity obtained
Colony diameter of control
from triplicate assays.
The inhibition of protein denaturation in percentage was calcu-
2.5.3.2. Mycelium dry weight method. Mycelium dry weight method lated from the following formula:
given by Sahayaraj et al. (2009) was used to observe the antifungal   
Sample ðAbsÞ
activity of bark of P. ciliata crude methanolic extract and various frac- % Inhibition ¼ 100 x
1
Control ððAbsÞ
tions. Test tubes containing PDA broth and BCE in different concen-
trations (0.4
1 mg/mL per test tube from the 20 mg/mL in 10%
DMSO stock solution) were inoculated with 6 mm diameter mycelial 2.6.2. Egg albumin (fresh) protein denaturation assay
fungal bit and incubated for 7 days at 25 °C. The visible growth of fun- In this assay, 0.2 mL of egg albumin was mixed with 2.8 mL of PBS
gal mycelium in the tubes expressed the extract’s degree of activity. (pH 6.4) in the test tubes. After this, 2 mL of each concentration
The fungal mycelium was separated from the test tube by Whatman (0.2
1 mg/mL) of BCE was separately added to the solution. After
filter paper and then allowed to dry at 60 °C until it reached a con- that, test tubes were incubated for 15 min at 37 °C and after that,
stant weight. The hygromycin-b (0.100 mg/mL in dH2O) was used as heat for 5 min at 70 °C. After this, absorbance was measured at
a positive control. The potato dextrose broth without extract was 660 nm. Diclofenac sodium (0.2
1 mg/mL) was used as reference
used as a control mycelium. A similar procedure was repeated sepa- drug (Banerjee et al., 2014). The results were recorded in triplicate.
rately with triplicate BCE-fractions, LCE, and LCE-fractions. The same procedure was repeated separately with triplicate BCE-
The formula calculated the percentage inhibition (%): fractions, LCE, and LCE fractions. The IC50 value was calculated from
Weight of control mycelium
Weight of extract mycelium the linear regression of plots of different test sample concentrations
% Inhibition ¼ X 100
Weight of control mycelium against the mean percentage inhibition of the anti-inflammatory
activity obtained from triplicate assays.
The inhibition of protein denaturation in percentage was calcu-
2.5.4. Minimum inhibitory concentration (MIC) lated from the following formula:
The microtiter broth dilution method given by the Clinical and Lab-   
Sample ðAbsÞ
oratory Standards Institute (CLSI, 2012) was used for evaluating MIC %Inhibition ¼ 100 X
1
Control ðAbsÞ
values for antibacterial and antifungal assays. The 100 mL of the bark
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250

2.7. Cytotoxic activity was noted for the yield percentage among the crude extract, ethyl
acetate, and butanol fractions of bark and leaves (Fig. 1A). Similar to
2.8.1. Cell culturing and maintenance the present study, various authors also used chloroform, ethyl ace-
The cytotoxicity assay was carried out by using the MTT assay tate, n-butanol, and methanol solvents for fractionation of phytocom-
(Kumari et al., 2021) against two human colon cancer cell lines (HCT- pounds from P. tremuloides stem bark (Diouf et al., 2009), P. tomentosa
116, SW-620) which were purchased from National center for Cell stem bark (Liu et al., 2012), and P. tomentosa male inflorescence
Sciences (NCCS), Pune, India. The stock cells were cultured in Dulbec- (Hou et al., 2021). The authors observed maximum yield in n-butanol
co's Modified Eagle Medium (DMEM) supplemented with 10% inacti- fraction as compared to other solvents. According to Stalikas (2007),
vated Fetal bovine serum (FBS) and penicillin-streptomycin (1%) in a chemical composition, solvent's polarity, nature of phytoconstituents,
humidified atmosphere of CO2 (5%) at 37 °C until confluent. The used types of plant parts, storage times, and temperature also affects the
media was disposed of and washed with PBS to separate the dead yield percentage.
cells and debris. The trypsin (0.25%) was used for cells dissociation
and the pellet was cultivated after the centrifugation process at 3.2. Quantification of polyphenolic contents
3000 rpm for 10 min. The fresh aliquots were made and transferred
to new culture dishes for sub-culturing. Polyphenolic compounds such as phenols and flavonoids are bio-
logically active plant constituents, with redox properties responsible
2.8.2. In-vitro cytotoxicity assay (MTT assay on HCT-116 and SW-620) for antioxidant activity. They are numerously present in the Populus
The cancer cells (HCT-116; SW-620) at a density of 1 £ 104 cells/ species, widely used in food products, and responsible for providing
mL were seeded in a 96-well plate and incubated for 24 h 37 °C under many health benefits (Devappa et al., 2015a;2015b). In the present
5% CO2. The incubated cancer cells in the next day were treated with study, higher content of phenols was observed in the bark of P. ciliata
each extract separately in different concentrations (0.025
0.200 mg/ as compared to the leaves. BEF showed the highest TPC
mL) for 24 h, followed by MTT assay (4 h incubation) to assess the via- (317 § 5.61 mg/g GAE) followed by BBF (291.82§ 3.04 mg/g GAE),
bility of cells. As positive and negative controls vincristine and DMSO, LEF (291.78 § 4.19 mg/g GAE), LBF (278.11 § 2.57 mg/g GAE)
respectively were used. DMSO (100 mL) was added to each well for whereas lowest was observed in LHF (37.83 § 2.51 mg/g GAE)
dissolving the purple-coloured complex. Using a microplate reader, (Fig. 1B). A similar trend was observed with TFC, where maximum
the optical density was noted at 595 nm (Varioskan LUX, Thermo Sci- content of flavonoids was observed in BEF (271.26 § 4.16 mg/g QUE)
entific). followed by LEF (237.41§ 4.57 mg/g QUE), BBF (230.54 § 3.45 mg/g
Cell viability was analyzed by using following formula: QUE), LBF (203.92 § 2.70 mg/g QUE) and so on. A significant differ-
ence (p<0.05) was observed for both TPC and TFC among all the sam-
Abs ðCÞ
Abs ðSÞ
% Cell cytotoxicity ¼ X 100 ples of bark and leaves (Fig. 1B and 1C). Similar to the present study,
Abs ðCÞ
various researchers also observed high TPC and TFC in the ethyl ace-
here, Abs (C) was the absorbance of the control solution and Abs (S) tate fractions of bark and leaves of P. davidiana (Zhang et al., 2006), P.
was the absorbance of the test solution. tremuloides (Diouf et al., 2009), P. alba, and Salix subserrata
For the calculation of IC50 value, test sample’s linear regression of (Tawfeek et al., 2019). The difference in the amount of polyphenolic
plots of different concentrations was used against the mean percent- content (phenolic and flavonoids) in different fractions could be due
age inhibition of the cytotoxicity assay obtained from triplicate to the nature of the more soluble phytoconstituents in ethyl acetate
assays. n-butanol solvents (mild polar solvents). On the other hand, n-hex-
ane and chloroform, are nonpolar solvents having very little extrac-
2.8. Statistical analysis tion capacity, therefore, showing fewer contents of polyphenols.
In today world, HPTLC has become one of the most reliable and
The data was recorded in triplicate; Mean and SEM were calcu- popular methods for the estimation of phytoconstituents in the plant
lated on MS EXCEL 2016 from separate experiments. The paired sam- extracts because of better resolution, smaller analysis interval, lesser
ple T-test was used to analyze the overall significant difference use of the solvent system, and ability to analyze a large number of
(p<0.05) among all fractions and crude extract for TPC, TFC, and anti- samples simultaneously (Gomathi et al., 2012). In the current study,
oxidants, anti-inflammatory analysis through SPPS 20 software. The HPTLC analysis also revealed the presence of phenols and flavonoids
coefficient correlation (r2) was used to find out the correlation among in the bark, and leaves extracts of P. ciliata (Fig. 5 and 6). The Rf value
total polyphenolic contents and antioxidant values [IC50 values of of the standards (gallic acid, salicin, and quercetin) was compared
DPPH, ABTS, and FRAP assays] through SPSS 20 software. with the Rf value of compounds separated from the P. ciliata bark and
leaves samples. The result showed higher phenolic compounds viz.,
3. Results and discussion salicin (4.30§0.01 mg/mg), gallic acid (65.49§0.02 mg/mg), and quer-
cetin (57.29§0.02 mg/mg) in BEF fraction compared to other extracts
3.1. Yield percentage and fractions (Table 2). In addition to this, the bark had higher poly-
phenolic contents among both plant parts than leaves (Fig. 5 and 6).
P. ciliata is known for its polyphenolic compounds (phenolic acids, Through HPLC, Saqib et al. (2021) quantified the phenolic compounds
phenolic glycosides, and flavonoids) that have been reported to pos- viz., p-coumaric acid (2335.35 mg/g), vanillic acid (27.92 mg/g), cate-
sess several biological activities such as antimicrobial, anti-inflamma- chin (1881.61 mg/g), and quercetin (560.81 mg/g) in the methanolic
tory, and anticancer (Greenaway and Whatley, 1991; Kumari et al., crude extracts of P. ciliata (mixture of stem and leaves). Literature
2016; Devappa et al., 2015a; 2015b; Kumari et al., 2020). In the pres- also showed the antimicrobial, antioxidant, anti-inflammatory, and
ent study, both bark and leaves crude methanolic extracts were cytotoxic activities of salicin, quercetin, and gallic acid
sequentially fractionated with various solvents according to their (Albrecht et al., 1990; Boots et al., 2008; Lima et al., 2016; Sabaa et al.,
increasing relative polarity [n-hexane (0.009), chloroform (0.259), 2017; Hashemzaei et al., 2017; Tawfeek et al., 2019).
ethyl acetate (0.228), and n-butanol (0.389)]. In all extract and frac-
tions of bark and leaves, the maximum yield (%) was obtained in LBF 3.3. Antioxidant activities
(51.42§0.80%) followed by BBF (47.06§1.34%), LEF (34.73§1.08%),
BEF (34.73§1.08), and the lowest was observed in LHF (12.53§0.92) Free radicals (reactive oxygen species) are the molecules that
and BHF (10.00§0.40%) (Fig. 1A). A significant difference (p<0.05) cause oxidative stress in the body and are responsible for
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250

Fig. 1. Yield percentage (A), total phenol (B), total flavonoid content (C) in different extract and fractions of stem bark and leaves of P. ciliata. [Here, CE-crude extract, HF-n-hexane
fraction, CF-chloroform fraction, EF-ethyl acetate fraction, BF-n-butanol fraction. Different superscripts (a-e for bark CE, HF, CF, EF, and BF; fractions; f-j for leaves CE, HF, CF, EF and
BF) on bars are showing significant (p<0.05) variation and same superscript at the bar represent non-significant variation (p>0.05) among crude extracts and their fractions].

cardiovascular and autoimmune diseases, inflammation, rheumatoid (Kuchukhidze et al., 2010), P. ussuriensis stem bark (Si et al., 2011), P.
arthritis, and cancer (Krishnaiah et al., 2011), whereas antioxidants euphartica leaves (Zheng et al., 2012), P. alba leaf, and shoot
are the molecules that protect the cells from free radicals. In the cur- (Tawfeek et al., 2019).
rent study, the free radical scavenging activity of P. ciliata crude
methanolic bark and leaves extract and different fractions were ana-
lysed through DPPH, ABTS, and FRAP assays and IC50 values (Table S1 3.4. Correlation analysis
and Fig. 2A-C). Results showed that as the concentrations of the
extracts and fractions increased from 20 mg/mL-100 mg/mL the free A negative correlation coefficient (r2) was observed with TPC, TFC,
radical scavenging activity (% inhibition) was also increased (Table and IC50 values of DPPH, ABTS, and FRAP assays among bioactive frac-
S1). Among all samples (crude extracts and fractions), lowest IC50 tions of bark and leaves (Table 3) which shows that when the pheno-
value (DPPH: 25.75§3.60 mg/mL; ABTS: 33.55§2.96 mg/mL; FRAP: lic and flavonoid contents are increased in extract/fraction, its IC50
70.39§1.75 mM of Fe equivalents) was observed with BEF followed values decreased and lower IC50 values proved the higher antioxidant
by BBF (DPPH: 29.85§ 3.22 mg/ml; ABTS: 38.79 § 2.18 mg/ml; FRAP activities of extract/fraction (Fidrianny et al., 2015; Ruslan et al.,
(79.84 § 2.22 mg/ml), whereas highest (DPPH: 88.34§1.60 mg/mL; 2018). A similar correlation was observed among total polyphenolic
ABTS: 97.25§1.20 mg/mL; FRAP: 165.77§3.09 mM of Fe equivalents) contents and antioxidant capacity of P. tremuloides (Diouf et al.,
was with LHF (Fig. 2A-C). The ascorbic acid (DPPH: 6.83§ 0.54 mg/ 2009), P. nigra flowers (Pasca et al., 2016), P. nigra wood extracts
mL, ABTS: 24.24§1.47 mg/mL; FRAP: 30.96§2.56 mM of Fe equiva- (Todaro et al., 2017), P. alba leaf and shoot (Tawfeek et al., 2019), P.
lents) was used as a positive control (Fig. 2A-C). In all antioxidant balsamifera buds (Stanciauskaite et al., 2021), and P. nigra buds
assays, a significant difference (p<0.05) was observed for IC50 analy- (Stanciauskaite et al., 2021). The present study also showed a positive
sis among the crude extracts and fractions of P. ciliata bark and leaves correlation between IC50 values of DPPH assays and ABTS assay
(Fig. 2A-C). whereas, FRAP showed a negative correlation with both DPPH and
The present study showed maximum antioxidant activity in the ABTS assays (Table 3). This could be due to the reason that both DPPH
BEF and BBF which could be due to the high amount of TPC and TFC and ABTS assays have a similar mechanism of action and both are
in this fraction. Bors and Michel (2002) also stated that phenols and based on electron and H atom transfer whereas FRAP assays is based
flavonoids are valuable plant constituents for the scavenging of free on only electron transfer reaction (Prior et al., 2005; Al-Laith et al.,
radicals because of phenolic hydroxyl groups. These results were sim- 2019; Shahinuzzaman et al., 2020). According to Al-Laith et al. (2019)
ilar to the results obtained by different authors from P. tremula leaves and Shahinuzzaman et al. (2020), the positive correlation between
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250

Fig. 2. In-vitro antioxidant potential [DPPH (A), ABTS (B), FRAP (C)] in terms of IC50 values in different extract and fractions of stem bark and leaves of P. ciliata. [Here, CE-crude
extract, HF-n-hexane fraction, CF-chloroform fraction, EF-ethyl acetate fraction, BF-n-butanol fraction, AA-ascorbic acid. Different superscripts (a-e for bark CE, HF, CF, EF, and BF;
fractions; f-j for leaves CE, HF, CF, EF and BF) on bars are showing significant (p<0.05) variation and same superscript at the bar represent non-significant variation (p>0.05) among
crude extracts and their fractions].

DPPH and ABTS assays could be due to the involvement of similar ampicillin was used as positive control [P. aeruginosa (0.010 mg/mL),
biologically active compounds in antioxidant activity. K. pneumoniae (0.008 mg/mL), S. aureus (0.007 mg/mL), E. coli
(0.010 mg/mL), and B. subtilis (0.007 mg/mL)] (Table 1). Against all
3.5. Antimicrobial activities tested bacterial strains, the lowest MIC value was observed with BEF
and BBF against P. aeruginosa (0.015 mg/mL). Kumari et al. (2020)
Results of antimicrobial activity thorough disk diffusion assay also reported antibacterial activities of methanol and chloroform
showed that as the concentration of the extracts increased from extracts of P. ciliata leaves against Gram's negative (E. coli and P. aeru-
0.4 to 1 mg/mL, the zone of inhibition (ZOI) was also increased ginosa; MIC 0.78 mg/mL) and Gram's positive (B. subtilis and S. aureus;
(Table S2 and Fig. S1
2). Among all extracts and fractions, maxi- MIC 1.56 mg/mL) bacteria. The various authors also evaluated the
mum ZOI was observed with ethyl acetate and n-butanol frac- antibacterial activity of Populus species against different bacterial
tions of bark and leaves whereas the minimum was observed species. For example, ethyl acetate fractions of P. nigra buds (MIC
with chloroform and hexane fractions. Both BEF and BBF showed 0.814 mg/mL) (Nassima et al., 2019), methanolic bud extract P. nigra
almost similar activity against E. coli and S. aureus whereas, for (MIC (0.50 mg/mL) (Vardar-Unlu et al., 2008), ethanolic bud resin
remaining bacterial strains the variation was very minute. Com- extract of P. nigra (MIC 125 mg/mL) (De Marco et al., 2017) have
pared to both plant parts extracts and fractions, the bark had shown good antibacterial activity against S. aureus, B. subtilis, and P.
higher antibacterial potential than leaves. Populus has hardly aeruginosa, respectively. All these studies proved the antibacterial
been studied for its antimicrobial properties. properties of Populus species. Among all extracts and fractions,
Nassima et al. (2019) reported the maximum antibacterial activity the bark ethyl acetate faction showed higher antibacterial activity
(ZOI) of ethyl acetate fraction of P. nigra and P. alba buds against due to the presence of a high amount of biologically active phe-
B. subtilis, Staphylococcus aureus. The crude methanolic extract of nolic compounds. The phenolic compounds have hydroxyl groups
P. ciliata leaves also exhibited antibacterial activity against B. sub- which interrupts the hydrophobic and hydrophilic interactions
tilis, E. coli, S. aureus, and P. aeruginosa (Kumari et al., 2020). within the bacterial plasma membrane, create pores, and also
MIC data further supported these results. In comparison to all inhibit the toxin produced by bacteria. The cytoplasmic leakage
extracts and fractions, BEF showed the lowest MIC value for P. aerugi- and inhibition of enzymes further lead to the death of the micro-
nosa (0.015 mg/mL) followed by K. pneumoniae and S. aureus organisms (Kumar and Pandey, 2013; Bouarab-Chibane et al.,
(0.031 mg/mL), and E. coli and B. subtilis (0.062 mg/mL) (Table 1). The 2019; Alvarez-Martinez et al., 2020).
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Table 1
Minimum inhibitory concentration (MIC) of crude methanolic extracts and fractions of P. ciliata bark and leaves against different
microbial strains.

Bacterial strains

MIC (mg/mL)

Plant parts Extract or fractions E.coli P. aeruginosa K. pneumoniae S. aureus B. subtilis

Bark CE 0.125 0.250 0.125 0.125 0.500

HF 1.00 1.00 0.500 0.500 1.00
CF 0.500 0.250 0.250 0.500 0.500
EF 0.0625 0.0156 0.0312 0.0312 0.0625
BF 0.125 0.0625 0.0625 0.0312 0.125

Leaves CE 0.250 0.500 0.250 0.250 0.500

HF 1.25 1.25 1.00 1.00 1.25
CF 1.00 0.500 0.500 1.00 1.00
EF 0.125 0.0312 0.0625 0.125 0.250
BF 0.250 0.125 0.125 0.0625 0.250

PC AMP 0.010 0.010 0.008 0.007 0.007

MIC (mg/mL)

Plant parts Extract or fractions Plant pathogenic strains yeast strains

Human pathogenic Non-pathogenic

F. oxysporum R. necatrix C. albicans C. glabrata C. auris S. cerevisiae

Bark CE 1.000 1.00 10 10 2.5 10

HF 2.00 2.5 ND ND ND ND
CF 1.25 1.25 ND ND ND ND
EF 0.125 0.250 5 5 2.5 5
BF 0.500 0.500 5 5 1.25 5

Leaves CE 1.25 1.25 >20 >20 >40 >20

HF 2.25 2.5 ND ND ND ND
CF 1.50 1.5 ND ND ND ND
EF 0.250 0.5 >20 >20 >40 >20
BF 1.00 1.00 >20 >20 >20 >10

PC HYG 0.007 0.015 ND ND ND ND

Here, CE: crude methanolic extract, BF: n-butanol fraction, EF: ethyl acetate fraction, CF: chloroform fraction, HF: n-hexane frac-
tion, PC: positive control, AMP: ampicillin, HYG: hygromycin-b. Values are expressed as mean § standard deviation (n = 3).

Table 2
HPTLC quantification of polyphenolic compounds in crude extracts and different fractions of P. ciliata bark and leaves.

Plant parts Extract or fractions Salicin (S) Gallic acid (GA) Quercetin (Q)
mg/mg
Bark CE 6.11§0.07a 21.71§0.07 a 18.42§0.01a
HF ND 7.43§0.07 ab,b 6.01§0.07 ab,b
CF ND 12.87§0.01 ac,bc,c 8.17§0.01 ac,bc,c
EF 4.30§0.01a,d 65.49§0.02 ad,bd,cd,d 57.29§0.02 ad,bd,cd,d
BF 4.65§0.02 a,d,e 42.93§0.02 ae,be,ce,de,e 15.81§0.01 ae,be,ce,de,e

Leaves CE 9.08§0.02 af,df,ef,f 17.16§0.01 af,bf,cf,df,ef,gf,f 7.21§0.01 af,bf,cf,df,ef,gf,f

HF ND 7.21§0.02 ag,bg,cg,dg,eg,hg,ig 3.34§0.02 ag,bg,cg,dg,eg,hg,ig
CF ND 11.50§0.01 ah,bh,ch,dh,eh,fh,ih,h 7.11§0.06 ah,bh,ch,dh,eh,f,ih,h
EF 5.87§0.06 a,d,eh,fh,h 45.06§0.01 ai,bi,ci,di,ei,fi,gi,hi,gi,hi,i 13.19§0.03 aj,bj,cj,dj,ej,fj,gj,hj,i
BF 5.91§0.08 a,d,ei,fi,h 37.77§0.01 aj,bj,cj,dj,ej,fj,gj,hj,ij 14.52§0.02 aj,bj,cj,dj,ej,fj,gj,hj,i,j
Here, CE-crude extract, HF-n-hexane fraction, CF-chloroform fraction, EF-ethyl acetate fraction, BF-n-butanol fraction. Differ-
ent superscripts (a-e for bark CE, HF, CF, EF, and BF; fractions; f-j for leaves CE, HF, CF, EF and BF) on bars are showing signifi-
cant (p<0.05) variation and same superscript at the bar represent non-significant variation (p>0.05) among crude extracts
and their fractions.

Similar to the antibacterial assays, the antifungal activity was also oxysporum (0.125 mg/mL) and R. necatrix (0.250 mg/mL) as compared
increased with an increase in the extract’s concentration from 0.4 to to other extracts and fractions of bark and leaves (Table 1). The
1 mg/mL (Table S3). For both fungi (F. oxysporum and R. necatrix) the hygromycin-b was used as positive control (MIC 0.007 mg/mL for F.
maximum percentage inhibition was observed with butanol fraction oxysporum and 0.015 mg/mL for R. necatrix) (Table 1). The methanolic
and minimum with hexane fraction (Table S3 and Fig. S3
6). Similar leaves extract of P. euphratica showed antifungal activity (MIC 0.
to the present study, Populus x euramericana leaves (Zhou et al., 390 mg/mL) against F. oxysporum (Khaleel, 2019).
2009) and P. nigra buds (Debbache-Benaida et al., 2013) also showed In all antimicrobial assays, the CE, BF, and EF of bark and leaves
higher percentage inhibition of Fusarium species. The MIC results also were potentially active against tested microorganisms. Therefore,
proved the maximum antifungal potential of the BFE against F. these two fractions of bark and leaves and crude extract were further
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250

Table 3
Pearson Correlation matric (r2) in between different IC50 values of invitro antioxidant activities and the total phenolics
or flavonoids content of crude extracts and different fractions of P. ciliata bark and leaves.

TPC TFC DPPH ABTS FRAP TPC TFC DPPH ABTS FRAP

BCE LCE

TPC 1 .985
0.282
0.653
0.720 1 .789
0.744
0.808 .799

TFC 1
0.113
0.513
0.590 1
0.796
0.820
0.725
DPPH 1 .911
0.869 1 .806
0.716
ABTS 1
0.996 1
1.000**
FRAP 1 1

BBF LBF

TPC 1 .998*
0.877
0.188
0.991 1 1.000**
0.822
0.857
0.879

TFC 1
0.643
0.532
0.972 1
0.830
0.866
0.893
DPPH 1 .306
0.805 1 .994
0.877
ABTS 1
0.818 1
0.839
FRAP 1 1

BEF LEF
**
TPC 1 1.000
0.783
0.775
0.614 1 .992
0.924
0.920
0.967
TFC 1
0.543
0.533
0.834 1
0.962
0.991
1.000*
DPPH 1 1.000**
0.811 1 .954
0.981
ABTS 1 .823 1
0.987
FRAP 1 1
CE: crude methanolic extract, BF: n-butanol fraction, EF: ethyl acetate fraction, CF: chloroform fraction, HF: n-hexane
fraction, Bark: B, Leaves: L.

used to observe the antifungal activity against the human pathogenic the alteration and destruction of hydrogen, electrostatic, covalent,
Candida species (C. albicans, C. glabarata, and C. auris) non-pathogenic and disulfide bonds of proteins through chemical and physical
fungi (S. cerevisiae) through MIC assay. Results showed the antifungal agents. Moreover, protein denaturation is a well-documented cause
activity of BBF and BEF for C. auris at a minimum concentration of for inflammation and inflammatory-related diseases like rheumatoid
1.25 mg/mL and 2.5 mg/mL, respectively in comparison to the other arthritis, diabetes, and cancer (Chandra et al., 2012; Saravanan et al.,
tested fungal strains (Table 1). Vardar-Unlu et al. (2008) reported the 2020). Additionally, when cells undergo stress due to various stimuli
antifungal activity of methanolic bud extract of P. nigra, P. alba, and P. (microbes, chemicals, and physical), they will result in inflammation
tremuloides against Candida spp. with MIC ranges from 0.50 to 1 mg/ and characterized by various symptoms like tissues redness, swelling,
mL. Similarly, the n-butanol and ethyl acetate fractions of Salix and pain (Lekouaghet et al., 2020). Natural compounds (secondary
mucronata leaves (4 mg/mL) (El-Sayed et al., 2019) and Casearia syl- metabolites) of the plants have the capability to prevent proteins
vestris leaves (125->250 mg/mL) (Pereira et al., 2017) also exhibited from denaturation, therefore, can be utilized in the development of
antifungal activity against Candida spp. All these data supported the anti-inflammatory drugs to reduce inflammations in the body
potent antifungal activity of the BEF and BBF against plants and (Yesmin et al., 2020). In the present study, both protein denaturation
human pathogenic fungi, respectively. assays (egg albumin and bovine albumin) were performed by using
different concentrations (0.2
1.0 mg/mL) of bark and leaves extract
3.6. In-vitro anti-inflammatory activity and fractions (Table S5). Results showed that the highest anti-inflam-
matory potential of BEF (IC50 0.113§0.017 mg/mL for egg’s albumin
Protein denaturation is a process that involves the loss of tertiary and 0.160§0.014 mg/mL for BSA) and lowest was observed with LHF
and secondary structure of the proteins (Banerjee et al., 2014) due to (IC50 1.54§ 0.006 mg/mL for egg’s albumin and 1.56 § 0.011 mg/mL

Fig. 3. In-vitro anti-inflammatory potential [A-egg albumin assy (EA), B-bovine serum albumin assay (BSA)] in terms of IC50 values in different extract and fractions of stem bark and
leaves of P. ciliata. [Here, CE-crude extract, HF-n-hexane fraction, CF-chloroform fraction, EF-ethyl acetate fraction, BF-n-butanol fraction, DSC-Diclofenac sodium. Different super-
scripts (a-e for bark CE, HF, CF, EF, and BF; fractions; f-j for leaves CE, HF, CF, EF and BF) on bars are showing significant (p<0.05) variation and same superscript at the bar represent
non-significant variation (p>0.05) among crude extracts and their fractions].

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250

Fig. 4. In-vitro cytotoxicity assessment in terms of IC50 values in different extract and fractions of stem bark of P. ciliata. [Here, CE-crude extract, HF-n-hexane fraction, CF-chloro-
form fraction, EF-ethyl acetate fraction, BF-n-butanol fraction, VIN-vincristine. Different superscripts (a-e for CE, HF, CF, EF, and BF in HCT-116 cell line; f-j for CE, HF, CF, EF, and BF
in SW-620 cell line)) on bars are showing significant (p<0.05) variation and same superscript at the bar represent non-significant variation (p>0.05) among crude extract and their
fractions].

Fig. 5. HPTLC quantification of salicin (S), gallic acid (GA) and quercetin (Q). Here, A- A-HPTLC fingerprint standards, B- TLC fingerprint of each standard (S, GA, Q) in different
amount (200
1500 ng/spot) visualized under UV light (254 nm), C-TLC fingerprint visualized under white light, D-Linear regression curves of standards. 1
10 representing the
spot used for different concentrations.

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250

Fig. 6. HPTLC quantification of salicin (S), gallic acid (GA) and quercetin (Q) in various extracts and fractions of stem bark and leaves of P. ciliata. [Here, A-HPTLC fingerprint of differ-
ent extracts and fractions (BCE-bark crude extract, BBF-bark butanol fraction, BEF-bark ethyl acetate fraction, BCF-bark chloroform fraction, BHF-bark n-hexane fraction, LCE-leaves
crude extract, LBF-leaves butanol fraction, LEF-leaves ethyl acetate fraction, LCF-leaves chloroform fraction, LHF-leaves n-hexane fraction), B- TLC fingerprint of various different
extracts and fractions, C- TLC fingerprint of various different extracts and fractions visualized under UV light (254 nm)].

for BSA) whereas diclofenac sodium (IC50 0.095 § 0.023 mg/mL for (IC50 0.147 § 0.015 mg/mL) followed by BEF (IC50 0.168 § 0.016 mg/
egg’s albumin and 0.101 § 0.002 mg/mL for BSA) was used as control mL) (Fig. 4), whereas, in the case of SW-620, BEF (IC50
(Fig. 3A, B). For both protein denaturation assays, a significant varia- 0.332 § 0.015 mg/mL) was observed as the most active fraction fol-
tion (p<0.05) was observed among CE, BF, EF fractions of bark and lowed by BCE (IC50 0.338 § 0.015 mg/mL). The vincristine was used
leaves and positive control. The ethyl acetate fraction of Salix caprea as positive control against both cell lines (IC50 0.022 § 0.015 mg/mL
flowers have shown similar anti-inflammatory activity (IC50 for HCT-116, 0.017 § 0.012 mg/mL for SW-620) (Fig. 4). A significant
0.183 mg/mL) through heat-induced assay (Ahmed et al., 2011). variation (p<0.05) was observed for both cell lines among IC50 values
None of the report is available in the literature on in-vitro anti- of BCE, BBF, BEF of bark fractions. Enayat et al. (2013) also observed
inflammatory assessment on Populus species through in-vitro protein similar cytotoxic activity results in Salix aegyptiaca bark where the
denaturation assays. Zimmermann and Curtis (2017) reported that highest activity was observed in methanolic extract (IC50240 § 3 mg/
some alterations in the structure of anti-inflammatory drugs can mL) followed by the ethyl acetate fraction (IC50313 § 11.4 mg/mL)
increase their antimicrobial potential. Some anti-inflammatory drugs against HCT-116 cell line. Saqib et al. (2021) studied the in-vivo acute
like acetaminophen, acetylsalicylic acid, and other non-steroidal toxicity and sub-acute toxicity of crude extract of mixture of P. ciliata
anti-inflammatory drugs (NSAIDs) have also been reported in the stem and leaves on rats and reported the safe concentration of the
control of bacterial and fungal strains growth, especially Candida spe- extract up to 3000 mg/kg. The maximum activity of the crude extract
cies (Zimmermann and Curtis, 2017). could be due to the cumulative effect of the bioactive compounds
present in the crude extract which get separated at the time of frac-
3.7. Cytotoxic activity tionation. Therefore, overall, the BEF was observed to be the most
biological active fraction as followed by BBF and LEF. Bark's higher
In all the assays, bark crude extracts and fractions (BEF and BBF) biological potentiation (antioxidant, antibacterial, antifungal, anti-
showed higher anti-inflammatory potential than the leaves crude inflammatory, and cytotoxic activities) validate its traditional medici-
extract and fractions. Therefore, bark extracts and fractions were nal uses.
used for cytotoxicity assessment. The cytotoxicity results also
showed a similar trend as observed with previous experiments, the 4. Conclusion
cytotoxic activity was increased with increase in extract concentra-
tion (0.025
0.2 mg/mL) (Table S4, Fig. S7
8). In the results highest This investigation is the first report on the comparative screening
anticancer potential for cell lines, HCT-116 was obtained with BCE of P. ciliata bark and leaves crude methanolic extracts and their
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I. Guleria, A. Kumari, M.-A. Lacaille-Dubois et al. South African Journal of Botany 148 (2022) 238
250

different fractions for various biological activities. The study revealed Chaudhary, A., Sharma, S., Mittal, A., Gupta, S., Dua, A., 2020. Phytochemical and antiox-
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Declaration of Competing Interest as value-added co-products from the wood and cellulosic bioethanol industry. Bio-
energy Res. 8, 1235–1251.
The authors declare that they have no conflict of interest. Diouf, P.N., Stevanovic, T., Cloutier, A., 2009. Antioxidant properties and polyphenol
contents of trembling aspen bark extracts. Wood Sci. Technol. 43, 457–470.
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