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The biological activities of roots and aerial parts of Alchemilla vulgaris L.
T. Boroja
a,
⁎,V.Mihailović
a
, J. Katanić
a
, S.-P. Pan
b
,S.Nikles
b
, P. Imbimbo
c
,D.M.Monti
c
, N. Stanković
a
,
M.S. Stanković
d
, R. Bauer
b
a
Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia
b
Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Universitaetsplatz 4/1, 8010 Graz, Austria
c
Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario Monte Sant'Angelo, via Cinthia 4, 80126 Naples, Italy
d
Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića12,34000Kragujevac,Serbia
abstractarticle info
Article history:
Received 14 December 2017
Received in revised form 9 March 2018
Accepted 20 March 2018
Available online xxxx
Edited by AO Aremu
Medicinalplants are considered to be a major source of biologically active compounds, which provides unlimited
opportunities for their use either as medical treatments or as novel drug formulations.
The focus of our study was on basic phytochemical analysis and in vitro examination of the biological activity of
Alchemilla vulgaris L. Methanolic extracts of above ground parts and roots of A. vulgaris (AVA and AVR, respec-
tively) were prepared by maceration for 72 h. Phytochemical profile of extracts was evaluated by spectrophoto-
metric determinations of phenolic compounds and HPLC-PDA analysis. AVA and AVR were analysed for their
antioxidant efficacy as total antioxidant capacity, metal chelation and reducing power ability, inhibition of lipid
peroxidation as well as their potential to neutralise DPPH, ABTS, and OH radicals. Microdilution method was
employed to investigate the antibacterial and antifungal activity of extracts against nine ATCC and isolates of
bacteria and ten fungal strains from biological samples. Anti-inflammatory activity of the extracts was evaluated
using cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)assays and the assayfor determinationof COX-2
gene expression, while biocompatibility of extracts was assessed by MTT assay.
Our results revealed the high amount of phenolic compounds in both extracts; especially they were rich in
condensed tannins. Ellagic acid and catechin were tentatively identified in AVA and AVR, respectively. Full
biocompatibility as well as remarkable bioactivity were observed for both extracts in all employed assays, so
our further investigations will be focused on the identification of active constituents in A. vulgaris and the molec-
ular mechanisms of their action.
© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Keywords:
Alchemilla vulgaris
Lady's mantle
Antioxidant activity
Antimicrobial activity
Biocompatibility
Anti-inflammatory activity
1. Introduction
Several lines of evidence support the hypothesis that secondary
metabolites from plants (e.g. flavonoids and phenolic acids) may play
an antioxidant role and diminish the adverse effects of an imbalance
between the production of enzymatic and non-enzymatic antioxidants
and overproduction of free radicals in oxidative stress (Hussein and
Khalifa, 2014). As a result of their antioxidant activity, either through
their reducing capacity or through potential influences on intracellular
redox processes, phenolic compounds manifest various beneficial
effects, including anti-inflammatory and anticancerogenic activities
(Han et al., 2007; Li et al., 2014).
Alchemilla vulgaris L. (Lady's mantle), an herbaceous perennial plant
belonging to the Rosaceae family, is widely spread across Europe and
Asia and commonly known in traditional medicine for treatment of
ulcers, wounds, eczema, and digestive problems as well as a remedy for
gynaecological disorders, such as heavy menstrual flow, menorrhagia
and dysmenorrhoea (Jarićet al., 2015; Masullo et al., 2015;
Ilić-Stojanovićet al., 2017). Alchemilla species have been reported to
exert a variety of biological activities, including antiviral, antioxidant,
antiproliferative, and antibacterial activity as well as healing effects on
cutaneous wounds in rats (Trouillas et al., 2003; Shrivastava and John,
2006; Filippova, 2017). Previous findings showed that aerial parts of
A. vulgaris comprise mostly phenolic compounds –a large amount of
tannins, phenolic acids (predominantly ellagic acid, gallic, and caffeic
acids), flavonoids (quercetin), and flavonoid glycosides (isoquercetin,
rutin, avicularin, and tiliroside) (Møller et al., 2009). To the extent of
our knowledge, there is a scarce literature on phytochemical profile and
biological activity of roots of A. vulgaris.
South African Journal of Botany 116 (2018) 175–184
Abbreviations: AVA, Alchemilla vulgaris aerial parts methanolic extract; AVR, Alchemilla
vulgaris roots methanolic extract; ROS, reactive oxygen species; COX-1, cyclooxygenase-1;
COX-2, cyclooxygenase-2; PGH
2
, prostaglandin H
2
; NSAIDs, non-steroidal anti-
inflammatory drugs; IL, interleukin; TNF-α,tumournecrosisfactorα; iNOS, inducible nitric
oxide synthase; NF-κB, thenuclear factor kappa-light-chain-enhancer of activated B cells;
DEX, dexamethasone.
⁎Corresponding author.
E-mail address: tatjanaboroja@gmail.com.(T.Boroja).
https://doi.org/10.1016/j.sajb.2018.03.007
0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
Reactive oxygen species(ROS) encompass a broad range of reactive
molecules triggering a multitude of ailments, such as rheumatoid
arthritis, cardiovascular disorders, neurological disease, and cancer
(Hitchon and El-Gabalawy, 2004; Valko et al., 2004; Melo et al., 2011).
Increased ROS generation has been described as one of the key factors
in the progression of inflammatory disorders (Mittal et al., 2014).
Cyclooxygenases (COX-1 and COX-2) are enzymes involved in the
inflammatory process and responsible for the conversionof arachidonic
acid into pro-inflammatory mediators, like prostaglandin H
2
(PGH
2
).
COX-1 is a constitutive enzyme expressed in almost all cells providing
homeostatic functions. Under normal conditions, COX-2 is unexpressed
in most cells, but its expression can be induced by inflammatory stimuli.
Hence, COX-2 is a major target for anti-inflammatory therapies. Since the
use of non-selective COX inhibitors (non-steroidal anti-inflammatory
drugs-NSAIDs) may lead to side effects, particularly evident in the
gastrointestinal tract (Jones et al., 2008), novel COX-2-specific agents,
with no or very little undesirable effects, are urgently needed.
The presented study was focused on assessment of biocompatibility,
antioxidant, antimicrobial, and anti-inflammatory activities of methano-
lic extracts of aerial parts and roots of Lady's mantle (A. vulgaris) and
their phytochemical profile as well.
2. Materials and methods
2.1. Chemicals and instruments
All spectrophotometric determinations were performed on UV–Vis
double beam spectrophotometer Halo DB-20S (Dynamica GmbH,
Dietikon, Switzerland). Ellagic acid, hyperoside, rutin and TRIS/HCl-
buffer were obtained from Carl Roth (Karlruhe, Germany), trolox,
epicatechin, catechin, gallic acid, vanillic acid, rutin, kaempferol, querce-
tin, DMSO (N99.98% purity), and formic acid from Sigma-Aldrich
(Deisenhofen, Germany), caffeic acid and Na
2
EDTA (Titriplex III) from
Merck KGaA (Darmstadt, Germany). HPLC-grade acetonitrile, water,
and trifluoroacetic acid were purchased from Merck (Darmstadt,
Germany). Resazurin was purchased from Acros Organics (Geel,
Belgium), while all other chemicals used in antimicrobial experiments
were purchased from Torlak Institute of Virology, Vaccines, and Sera
(Belgrade, Serbia). Reagents used in COX-1 and -2 assays: purified
prostaglandin H synthase (PGHS)-1 from ram seminal vesicles, human
recombinant PGHS-2, NS-398, and arachidonic acid were obtained
from Cayman Chemical Co. (Ann Arbor, MI, USA), hematin (porcine)
and indomethacin from ICN (Aurora, Ohio, USA), epinephrine hydrogen
tartarate from Fluka (Buchs, Switzerland), and competitive PGE
2
EIA kit
from Assay Designs Inc. (Ann Arbor, MI, USA). COX-2 gene expression
kits, reagents and chemicals for this method: fetal bovine serum (FBS),
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), phos-
phate buffered saline (PBS), penicillin, and streptomycin human
leukemic monocytic cell line THP-1 (European Collection of Cell Culture;
Item No. 88081201), and RPMI1640 were obtained from Gibco® (NY,
USA), phorbol 12-myristate 13-acetate (PMA), while dexamethasone,
lipopolysaccharide (LPS) as well as GenElute™Mammalian TotalRNA
Miniprep Kit were from Sigma-Aldrich (MO, USA). High-Capacity
cDNA Reverse Transcription Kit, Pre-developed TaqMan® Assay,
COX-2 primers and COX-2 probe were from Applied Biosystems (NY,
USA). BalbC-3 T3 fibroblasts (clone A31) were purchased from ATCC
(Manassas, VA) and human epidermal keratinocytes (HaCaT) from
Innoprot (Biscay, Spain).
2.2. Plant material and preparation of the extracts
The roots and aerial parts of Alchemilla vulgaris L. were collected in
August 2014 at GočMountain (Central Serbia). Collection of plant
material was carried out by the sampling of 20 representative indi-
viduals of the population in the full flowering period. The taxonomic
and botanical identity was confirmed by Milan Stanković,PhD.A
voucher specimen (No. 120/015) is kept in the Herbarium of the
Department of Biology and Ecology, Faculty of Science, University of
Kragujevac, Kragujevac, Serbia. The collected plants were air-dried in
darkness at ambient temperature. The dried aerial parts and roots
separately from all individuals were cut up, mixed and powdered for
extracts preparation.
Dried and powdered aerial parts (30.00 g) and roots (55.80 g) of
A. vulgaris were macerated with methanol (150 and 280 mL, respec-
tively) for 24 h three times, by continuous stirring at room temperature.
Mass of the extracts was determined after filtration through Whatman
No.1 filter paper and concentrating under reduced pressure at 45 °C.
The obtained dry weights of the extracts were 3.70 g for A. vulgaris aerial
parts (AVA) and 9.00 g for roots (AVR), (12% w/w and 16.13% w/w,
respectively). The obtained extracts were kept at +4 °C until further use.
2.3. Chromatographic analysis
The identification of individual phenolic compounds in the extracts
was performed using HPLC system (Shimadzu Prominence, Kyoto,
Japan) as described previously (Mihailovićet al., 2016). The detection
wavelength of PDA was monitored at 260, 280, 325 and 330 nm.
Methanolic solutions of ellagic acid, caffeic acid, gallic acid, vanillic
acid, quercetin, rutin, kaempferol, (+)-catechin, and (−)-epicatechin
were used as reference standards for the identification of compounds
in the extracts, which was performed by comparing retention times
and absorption spectra of the peaks with reference standards. The com-
pounds identified in the extracts were confirmed by spiking the sample
with the standard compound.
2.4. Phytochemical analysis
2.4.1. Total phenolics
The method developed by Singleton et al. (1998) was used to
determine the total phenolic content. 0.5 mL aliquots of the extracts
diluted in methanol were mixed with 2.5 mL of Folin–Ciocalteu solution
(previously diluted ten-fold with water) and 2 mL of 7.5% aqueous
NaHCO
3
solution. The reaction mixture was incubated for 15 min at
45 °C. The absorbance was read at 765 nm. Mass concentrations of
total phenols in plant material were determined using the standard
curve for gallic acid and results were calculated as gallic acid equiva-
lents (mg GAE/g dry weight of extract).
2.4.2. Total flavonoids
The total flavonoid content was estimated by the method of
Quettier-Deleu et al. (2000). The reaction mixture contained 0.5 mL 2%
AlCl
3
in methanol and 0.5 mL of extracts solutions in methanol
(1 mg/mL). The absorbance was measured at 415 nm after one hour of in-
cubation at room temperature. Results were calculated as milligrammes
of rutin equivalents per gram of dry weight of extract (mg RUE/g dry
weight of extract).
2.4.3. Phenolic acids
Determination of the total phenolic acids content in plant extracts
was performed according to the method described in the The Polish
Pharmacopoeia VIII (2009), with slight modifications. Briefly, 1 mL of
plant extracts solutions was added to 5 mL of distilled water, followed
by addition of 1 mL of 0.1 M HCl, 1 mL of Arnow's reagent (10%
Na-molybdate and 10% Na-nitrite), 1 mL of 1 M NaOH, and 1 mL of
distilled water. The absorbance was read immediately at 490 nm.
Results are presented as caffeic acid equivalents (mg CAE/g dry weight
of extract).
2.4.4. Determination of tannins
The method suggested by Scalbert et al. (1989) was used to estimate
the content of condensed tannins in plant extracts. In brief, the extracts
were mixed with a certain amount of phloroglucinol (for each equivalent
176 T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
of gallic acid in extracts 0.5 mol phloroglucinol was added). Subsequently,
1 mL of 4.8 M HCl solution and 1 mL of formaldehyde (13 mL of 37% form-
aldehyde diluted to 100 mL in water) were added. The reaction mixture
was allowed to stand overnight at room temperature to precipitate the
tannins. Total phenolics were determined in the solution above the pre-
cipitate using Folin–Ciocalteu method and this value was subtracted
from the total phenolics' value to obtain the total tannin content,
expressed as gallic acid equivalents (mg GAE/g dry weight of extract).
The gallotannin content was determined according to the procedure
described by Haslam (1965). To 1.5 mL of a saturated KIO
3
solution,
3.5 mL of a methanol solution of the examined extracts were added.
The absorbance of the red intermediate was sprectrophotometrically
determined at 550 nm until the maximum absorbance was reached.
The gallotannin content was determined as gallic acid equivalents
(mg GAE/g dry weight of extract).
2.4.5. Total anthocyanins content
Determination of total and monomeric anthocyanins was conducted
using single pH and pH differential methods (Cheng and Breen, 1991),
based on the ability of anthocyanins to change their structure depending
onthepH.Thespecified volume of the sample was mixed with pH 1.0 KCl
-buffer (0.025 M) and pH 4.5 sodium-acetate buffer (0.4 M), respectively.
After 30 min incubation, the absorbance was measured spectrophoto-
metrically at 520 and 700 nm. The concentrations of the total and
monomeric anthocyanins were determined as cyanidin-3-glycoside
equivalents according to the following equation: c = (A ∗M∗F∗1000) /
(ε∗l), where c - concentration of total or monomeric anthocyanins; A -
absorbance of total and monomeric anthocyanins, which is calculated
as (A
520
-A
700
)
pH 1.0
and (A
520
-A
700
)
pH 1.0
-(A
520
-A
700
)
pH 4.5
, respectively;
M - molar weight of cyanidin-3-glycoside (449.2 g/mol); F - dilution
factor; ε-molarabsorptivity(26900L/mol∗cm); l –cell length (1 cm).
2.5. Antioxidant activity
2.5.1. ABTS
·+
radical scavenging activity
Radical scavenging activity against ABTS radical cation (2,2′-azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid)) was measured spectrophoto-
metrically following the procedure of Re et al. (1999). The percentage
of ABTS
·+
decoloration is proportional to the ability of extract to neu-
tralise radicals and has been calculated using the formula: % inhibition =
((Ac −As) ∗100) / Ac, where: Ac –absorbance of the control (methanol
instead of the sample); As –absorbance of the sample.
The concentration of samples providing 50% of radical scavenging ac-
tivity (IC
50
) was calculated using dose–response sigmoidal curve plotted
the percentage of inhibition against extract concentration (μg/mL).
2.5.2. DPPH
·
radical scavenging activity
The ability of plant extracts to neutralise DPPH
·
radical was esti-
mated according to Kumarasamy et al. (2007). The reaction mixture
containing 1 mL of DPPH
·
solution in methanol (80 μg/mL) and 1 mL
of each extract solution (serial dilutions in methanol, started from
0.25 mg/mL) was allowed to stand in the dark for 30 min. The absor-
bance was read spectrophotometrically at 517 nm and IC
50
values
were calculated.
2.5.3. Hydroxyl radical scavenging activity
OH radical scavenging activity of extracts was determined using the
method performed by Kunchandy and Rao (1990).Briefly, 200 μLofthe
extract was mixed with 200 μL of 10 mM iron (III) chloride solution,
followed by 100 μL of 1 mM ascorbic acid solution, 100 μLof1mM
EDTA solution, 200 μL of 10 mM 2-deoxy-ribose solution, and 100 μL
of 10 mM hydrogen-peroxide solution. The reaction mixture was incu-
bated at 37 °C for 1 h. Subsequently, 1 mL of TCA-TBA solution (0.5%
TBA in 10% TCA water solution) was added and the final mixture was
incubated at 80 °C for 30 min and cooled to room temperature. The
absorbance of the cooled reaction mixtures was measured at 535 nm.
On the basis of the obtained absorbance values, the percentage of
inhibition and IC
50
values were calculated.
2.5.4. Estimation of metal chelating ability
The assessment of ability of plant extracts to inhibit the formation of
Fe
2+
-ferrozine complex was carried out according to the method by
Chew et al. (2009). One mililitre of 0.125 mM iron (II) sulphate solution
and 1 mL of 0.3125 mM ferrozine water solution were addedto 1 mL of
serial dilutions of extracts dissolved in methanol. The reaction mixture
then allows standing at room temperature for 10 min. The IC
50
values
were determined after reading the absorbance at 562 nm.
2.5.5. Reducing power
According to the method of Oyaizu (1986), to 2.5 mL of extracts
solutions in methanol (0.5 mg/mL), 2.5 mL of sodium-phosphate buffer
(0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide were added.
The reaction mixture was left to stand for 20 min at 50 °C, followed by
addition of 2.5 mL of 10% TCA. In 5 mL of this solution, 1 mL of 1% iron
(III) chloride solution was added and absorbance was read promptly at
700 nm. Trolox, as a referent antioxidant, was used for the construction
of calibration curve and the results of reducing capacity of tested extracts
were expressed as Trolox equivalents (mg TE/g dry weight of extract).
2.5.6. Total antioxidant activity
Prieto et al. (1999) developed a method for the determination of
the total antioxidant activity of plant extracts. In brief, 0.3 mL of the
extracts dissolved in methanol was mixed with 3 mL of reagent solu-
tion (0.6 M sulphuric acid, 28 mM sodiumphosphate, 4 mM ammonium
molybdate) and incubated for 90 min at 95 °C. Then, the reaction
mixture was cooled to room temperature and the absorbance of the
green-phosphate/Mo (V) complex was monitored at 695 nm. The
results are expressed as mg ascorbic acid (AA) per gram of dry extract,
using a standard curve for ascorbic acid.
2.5.7. Oil-in-water emulsion
Inhibitory activity of AVA and AVR towards lipid peroxidation was
performed according to the procedure described by Hsu et al. (2008).
To 0.5 mL of the serial dilutions of the extracts in methanol, 2.5 mL of
linoleic acid emulsion (0.2804 g of linoleic acid and 0.2804 g of
Tween-40 in 50 mL of 40 mM sodium phosphate buffer pH 7.0) was
added. The emulsion was incubated for 72 h at 37 °C. Thereafter,
0.1 mL of this solution was mixed with 4.7 mL of ethanol, 0.1 mL of
30% ammonium thiocyanate solution, and 0.1 mL of 20 mM iron (II)
chloride solution. Subsequently, the mixture was stirred for 3 min
and afterwards the absorbance was read spectrophotometrically at
500 nm against the methanol (blank).
2.6. Antimicrobial activity
2.6.1. Test microorganisms
The antimicrobial properties of A. vulgaris were tested against nine
bacterial and ten fungal strains. The employed bacteria were as follows:
Micrococcus lysodeikticus (ATCC 4698), Enterococcus faecalis (ATCC
29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC
70063), Pseudomonas aeruginosa (ATCC 10145), Salmonella typhimurium
(ATCC 14028), Bacillus subtilis (ATCC 6633), and isolated strains
from biological samples Bacillus mycoides (FSB 1) and Azotobacter
chroococcum (FSB 14). Antifungal activity was evaluated against ATCC
cultures of Aspergillus brasiliensis (ATCC 16404) and yeast Candida
albicans (ATCC 10259), whereas following fungi were isolated from bio-
logical samples: Phialophora fastigiata (FSB 81), Penicillium canescens
(FSB 24), Trichoderma viride (FSB 11), Trichoderma longibrachiatum
(FSB 13), Aspergillus glaucus (FSB 32), Fusarium oxysporum (FSB 91),
Alternaria alternata (FSB 51), and Doratomyces stemonitis (FSB 41). The
isolates of the bacteria and fungi were obtained from the Laboratory
for Microbiology, Faculty of Science, University of Kragujevac, Serbia.
177T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
The ATCC strains were provided from Institute of Public Health,
Kragujevac, Serbia.The bacteria and fungi cultures were subcultured
prior to testing; bacterial strains were cultured for 24 h at 37 °C on nutri-
ent agar (NA), C. albicans was cultured on Sabouraud dextrose broth
(SDB) for 24 h at 35 °C, whereas fungi were grown on potato glucose
agar (PDA).
2.6.2. Microdilution method
Microdilution method described by Sarker et al. (2007) was
employed to evaluate the antimicrobial activity of the samples, with
some modifications. Briefly, overnight-cultured bacterial cultures were
suspended in small amount of 5% DMSO than adjusted to the 0.5
McFarland turbidity standard using sterile normal saline and diluted to
obtain inoculum concentration of 5 × 10
6
CFU/mL for broth microdilution
MIC testing (CLSI, 2012). The analysed extracts (40 mg/mL), ellagic acid
and catechin (1 mg/mL) and antibiotic erythromycin (40 μg/mL) were
also dissolved in 5% DMSO. Determination of minimum inhibitory con-
centrations (MIC) of extracts for bacteria was performed in sterile 96
well plates (Spektar, Čačak, Serbia). 50 μL of two-fold serial diluted
extracts in Muller-Hinton broth (MHB) was added to each well, followed
by addition of 10 μL of resazurin (indicator), 30 μLofMHB,and10μLof
bacteria suspension. The final bacterial concentration of in each well
was 5 × 10
5
CFU/mL (CLSI, 2012). Each plate also included positive
(erythromycin at a concentration range 20–0.156 μg/mL), growth
(MHB, resazurin, and bacteria suspension) and sterility (MHB and
resazurin, without bacteria suspension) controls. The microplates were
incubated for 24 h at 37 °C. The lowest concentration of the extracts
containing blue-purple indicator's colour was considered as MIC.
Fungal species were cultured on PDA at 28 °C from 48 h to 5 days.
Obtained colonies covered with a small volume of 5% DMSO to obtain
the suspension, and then a final concentration of inoculum suspension
was adjusted with sterile normal saline to 5 × 10
4
CFU/mL in accordance
with NCCLS recommendation (NCCLS, 2002a, 2002b). The concentra-
tion of the extracts was 40 mg/mL, 40 μg/mL for antimycotic nystatin,
and 2 mg/mL for ellagic acid and catechin. MICs for fungal species
were also determined in sterile 96 well plates (NCCLS, 2002a, 2002b).
50 μL of serially diluted extracts in SDB, 40 μLofSDB,and10μLoffungal
suspension were added to each well, whereupon microplates were
incubated at 28 °C for 48 h. MICs were determinedas the lowest concen-
tration of extracts without visible fungal growth.
2.7. Evaluation of anti-inflammatory activity
2.7.1. COX-1 and COX-2 in vitro assays
The inhibition of COX-1 and COX-2 enzymes were evaluated using
in vitro assays in a 96-well plate with prostaglandin H synthase
(PGHS)-1 from ram seminal vesicles for COX-1 and human recombinant
PGHS-2 for COX-2 as previously described (Fiebich et al., 2005) with
modifications published by Katanićet al. (2016).Briefly, 10 μLof
extracts (50 μg/mL) dissolved in DMSO were added to the incubation
mixture containing 180 μL of 0.1 M TRIS/HCl-buffer (pH 8.0), 5 μM
hematin, 18 mM epinephrine hydrogen tartarate, 0.2 U enzyme
preparation and 50 μMNa
2
EDTA (only for COX-2 assay) and allowed
to stand for 5 min. Positive controls, indomethacin (1.25 μM, for
COX-1) and NS-398 (5 μM, for COX-2) were also dissolved in DMSO.
To start the reaction, 10 μLof5μM arachidonic acid in ethanol was
added to the reaction mixture. After 20 min of incubation at 37 °C, the
reaction was terminated by adding 10 μL of 10% formic acid.
The competitive PGE
2
EIA kit was applied for the determination of
the PGE
2
, the main arachidonic acid metabolite in this reaction. The
microplate reader (Tecan Rainbow, Switzerland) was used for evalua-
tion of EIA and the PGE
2
concentration was determined according to
the method described by Fiebich et al. (2005). All experiments were
performed in at least three independent experiments run in duplicate.
Inhibition of COX refers to the reduction of PGE
2
formation in compari-
son to a blank without inhibitor.
2.7.2. COX-2 gene expression assay
COX-2 gene expression analysis was performed in accordance with
the previously described method (Livak and Schmittgen, 2001) and
with slight modifications described in Katanićet al. (2016). The differen-
tiated human leukemic monocytic cell line THP-1 were treated with
plant extracts (25 μg/mL) for 1 h and stimulated with 7.5 ng/mL final
concentration LPS (lipopolysaccharide). Cells treated with DMSO
(dimethylsulfoxide ⩽0.1%) were used as calibrator sample.
2.8. Biocompatibility of extracts
BalbC-3 T3 fibroblasts and human epidermal keratinocytes were
cultured in Dulbecco's Modified Eagle's Medium, supplemented with
10% fetal bovine serum, 2 mM L-glutamine and antibiotics (streptomycin
and penicillin) in a 5% CO
2
humidified atmosphere at 37 °C.
For biocompatibility experiments, cells were seeded in 96-well plates
at a density of 2 × 10
3
/well (HaCaT cells) and 3 × 10
3
/well (BalbC-3 T3
cells). 24 h after seeding, increasing concentrations of the extracts (from
10 to 50 μg/mL) were added to the cells. After 48 and 72 h incubation,
cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay, as described by Del Giudice
et al. (2015). Cell survival was expressed as the percentage of viable
cells in the presence of the extract compared to controls. Two groups of
cellswereusedasacontrol,i.e. cells untreated with the extract and cells
supplemented with identical volumes of buffer. The average of the two
control groups was used as 100%. Each sample was tested in three inde-
pendent analyses, each carried out in triplicates.
2.9. Statistical analysis
The standard deviation was calculated using Microsoft Office Excel
2007 software and all results were expressed as mean values ± SD.
Statistical analysis of the results was performed by one-way analysis
of variance(ANOVA) using the OriginPro8 software package (OriginLab,
Northampton, Massachusetts, USA) for Windows. The statistical signifi-
cance was set at pb0.05.
3. Results
3.1. Phytochemical results
We analysed the total phenolic compounds by spectrophotometry
and HPLC-PDA to identify major phenolic components in the A. vulgaris
extracts. Our results of the analysis of phenolic compounds in methanolic
extracts of aerial parts and roots of A. vulgaris revealed a high content in
Table 1
Total phenolic compounds in methanolic extracts of aerial parts and roots of A. vulgaris.
mg GAEs/g d.w. mg RUEs/g mg CAEs/g mg C3Gs/g
Plant extracts Total phenolics Condensed tannins Gallotannins Total flavonoids Phenolic acids Total anthocyanins Monomeric anthocyanins
AVA 558.19 ± 4.83
a
386.70 ± 6.82
c
97.98 ± 0.01
e
13.30 ± 1.69
f,g
33.43 ± 1.15
h
8.41 ± 0.17
f,g,j
8.00 ± 0.18
f,j
AVR 442.32 ± 22.31
b
360.88 ± 2.17
d
n.d. 19.80 ± 0.35
g
57.36 ± 5.18
i
1.36 ± 0.06
j
0.95 ± 0.07
j
Results are expressed as mean values ± SD from three measurements; GAEs –gallic acid equivalents, RUEs –rutin equivalents, CAEs –caffeic acid equivalents, and C3Gs –cyanidine-3-
glycoside equivalents per gram of dry weight of extract; n.d. –not detected; means with different symbol in superscript are significantly different at pb0.05.
178 T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
both extracts. As can be seen from Table 1, spectrophotometrical deter-
mination demonstrated significantly higher (pb0.05) amount of total
phenolics in AVA (558.27 mg GAEs/g) in comparison with AVR
(442.32 mg GAEs/g). The most dominant phenolics in AVA were con-
densed tannins (proanthocyanidins) and gallotannins (386.70 and
97.80 mg GAEs/g), while AVR contained a slightly lower amount of con-
densed tannins (360.88 mg GAEs/g) and no detectable amount of
gallotanins. On the contrary, the concentration of hydroxycinnamic
acids derivatives was found to be significantly higher in the roots of
A. vulgaris, while no significant difference in the content of flavonoids
was observed. Anthocyanins content in the root extract was significantly
lower (pb0.05) when compared to all other examined classes of pheno-
lic compounds. Fig. 1 presents HPLC-PDA chromatograms of AVA and
AVR. Two of the peaks were tentatively identified as ellagic acid in the
above ground parts and (+)-catechin in the roots extract by matching
their retention times and comparing their UV-spectra at 260, 280, 325
and 330 nm. The identified compounds in the extracts were also con-
firmed by spiking the extracts with reference compounds.
3.2. Antioxidant activity
Becauseof the drawbacks of the individual use ofany in vitro method
for evaluation of antioxidant activity, severalmethods for screeningthe
antioxidant potential of AVA and AVR have been employed and the re-
sults are reported in Table 2. The absorbance of green phosphate/Mo
(V) complex formed atacidic pH was measured to evaluate the total an-
tioxidant activity of the extracts. The obtained results showed that AVR
exerts a higher total antioxidant activity than AVA (316.5 and 265.6 mg
ascorbic acid/g, respectively). The potential ability of the extracts to
neutralise free radicals was investigated using DPPH
·
,ABTS
·+
,and
·
OH assays. In these assays, AVA showed better antiradical activity
with significantly lower (pb0.05) IC
50
values than AVR. Considering
the reference antioxidants, AVA and AVR could scavenge DPPH
·
,
ABTS
·+
, and
·
OH radicals at significantly lower (pb0.05) concentra-
tions than the synthetic antioxidant butylated hydroxytoluene (BHT).
Furthermore, there was no statistically significant difference in DPPH
·
radical scavenging activities between AVA andthe well-known phenolic
Table 3
Antibacterial activity of methanolic extracts of aerial parts androots of A. vulgaris.
Bacterial species MIC values
a
AVA AVR Ellagic acid Catechin Erythromycin
Micrococcus lysodeikticus (ATCC 4698) 0.156 0.156 0.5 N120
Salmonella typhimurium (ATCC 14028) 0.625 0.625 0.5 0.5 20
Bacillus subtilis (ATCC 6633) 2.5 1.25 0.25 0.5 10
Enterococcus faecalis (ATCC 29212) 0.625 0.156 0.031 N1 1.25
Escherichia coli (ATCC 25922) 1.25 1.25 N1N15
Klebsiella pneumoniae (ATCC 70063) 5 10 N1N1N20
Pseudomonas aeruginosa (ATCC 10145) 2.5 5 1 1 20
Bacillus mycoides (FSB 1) 0.625 0.156 0.016 N1 1.25
Azotobacter chroococcum (FSB 14) 5 2.5 0.031 N120
a
MIC values, minimum inhibitory concentrations given as mg/mL for plantextracts, ellagic acid, and catechin, and as μg/mL for antibiotic erythromycin.
Table 2
Antioxidant capacity of the A. vulgaris aerial parts and roots methanolic extracts andstandards: BHT, catechin, and ellagic acid.
Plant extracts and standards IC
50
value (μg/mL) Total antioxidant
activity
(mg AA/g of extract)
Reducing capacity
(mg Trolox/g of extract)
Radical scavenging activity Inhibition of lipid peroxidation
DPPH•ABTS
+
•OH Oil-in-water system
AVA 5.96 ± 0.21
a
14.80 ± 2.15
a
13.06 ± 0.97
a
31.91 ± 3.12
a
265.62 ± 12.10 632.99 ± 10.26
AVR 11.86 ± 0.56
b
32.49 ± 1.95
b
18.44 ± 1.11
b
475.13 ± 11.41
b
316.47 ± 18.71 607.52 ± 10.01
BHT 26.25 ± 1.9
c
44.67 ± 3.00
c
21.93 ± 0.47
c
4.53 ± 0.07
c
n.t. n.t.
Catechin 7.52 ± 0.04
a
5.97 ± 0.16
d
6.32 ± 0.33
e
6.63 ± 0.11
c
n.t. n.t.
Ellagic acid 3.54 ± 0.13
d
8.14 ± 0.18
d
7.18 ± 0.89
e
10.87 ± 0.50
c
n.t. n.t.
AA –ascorbic acid; means in the same column with different symbol in superscript are significantly different at pb0.05.
Fig. 1. HPLC-PDA chromatogram of A. vulgaris methanolic extract ofaerial parts (A) and roots (B) recorded at 280 nm with tentatively identified ellagic acid (1) and (+)-catechin (2).
179T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
antioxidant catechin. The significant difference (pb0.05) in antioxidant
activity between the two extracts was also observed in an oil-in-water
system, where AVR showed almost fifty times higher IC
50
value than
AVA (475.1 and 31.9 μg/mL, respectively). Quite similar results for
AVA and AVR were obtained in the reducing power assay (633.0 and
607.5 mg Trolox/g, respectively).
The ferrous ion chelating test was employed to estimate the ability of
the extracts to chelate transition metals and to avoid the iron-overload
and generation of free radicals. All tested samples failed to chelateFe
2+
at concentration 1 mg/mL.
3.3. Antimicrobial activity
The results obtained for antimicrobialactivity are shown in Tables 3
and 4.Enterococcus faecalis,Salmonella typhimurium, Micrococcus
lysodeikticus, and Bacilus mycoides were the most sensitive examined
bacterial species to the tested A. vulgaris extracts, with MICs between
0.156 and 0.625 mg/mL.On the contrary, Klebsiella pneumoniae was
the most resistant bacteria in our study (MIC = 5 mg/mL for AVA and
10 mg/mL for AVR). MICs values above 1 mg/mL for AVA and AVR
were also observed for Pseudomonas aeruginosa, Bacillus subtilis,
Azotobacter chroococcum,andEscherichia coli. Ellagic acid and catechin
were used as reference compounds. Catechin failed to inhibit the
growth of all tested bacteria at concentrations lower than 0.5 mg/mL.
We observed that ellagic acid has antibacterial potential against the
same bacteria as the extracts, but MICs obtained for it were lower
than those for the extracts for the majority of bacteria. The commer-
cially available antibiotic erythromycin was more active against all
tested bacteria than the investigated extracts, ellagic acid, and catechin,
with MICs ranging from 1.25 to 20 μg/mL.
The investigated extracts showed similar activity against most of the
tested fungi, with MICs from 2.5 to above 20 mg/mL (Table 4). AVA and
AVR exhibited negligible antifungal activity against Doratomyces
stemonitis (2.5 and 5 mg/mL, respectively) and Aspergillus glaucus (5
and 10 mg/mL, respectively). On the contrary, AVA failed to inhibit
both Trichoderma species, while AVR did not show any antifungal effect
against Aspergillus brasiliensis and Alternaria alternata at a concentration
of 20 mg/mL.Moreover, both extracts did not inhibit the growth of Can-
dida albicans at the highest tested concentration.Ellagic acid failed to in-
hibit the growth of the majority of the employed fungi at a
concentration lower than 1 mg/mL, except for P. canescens (MIC =
0.25 mg/mL), while catechin did not exhibit any antifungal activity at
the same concentration. Our results showed the lower activity of AVA,
AVR, and reference compounds against all tested fungi in comparison
with the antimycotic nystatin, which has demonstrated antifungal
activity at concentrations from 0.078 up to 5 μg/mL.
3.4. Anti-inflammatory activity
Fig. 2. represents results of COX-1 and COX-2 inhibition assays as
well as COX-2 gene expression. Our results revealed that at a concentra-
tion of 50 μg/mL, AVA was capable of inhibiting the activity of COX-1
enzyme by 44.4%, whereas the inhibition of COX-2 was higher
(63.6%). Similar results were observed for AVR (44.1% for COX-1 and
40.4% for COX-2). The tested extracts at a concentration of 25 μg/mL
did not inhibit COX-2 gene expression.
3.5. Biocompatibility results
Finally, we analysed the biocompatibility of the extracts by perfor ming
a cell survival assay. The extracts were tested on immortalised murine
BalbC-3 T3 fibroblasts and human normal HaCaT keratinocytes in a
dose- and time-response test. As shown in Fig. 3, no significant differences
in cell survival between control group and groups treated with extracts
were observed. Indeed, both extracts showed total biocompatibility
with the two cell lines after 48 and 72 h.
4. Discussion
Notwithstanding that species from genus Alchemilla have been used
for many years in traditional medicine and are widely spread across
Europe, only a few reports have focused on their chemical composition
analysis. We tentatively identified ellagic acid in AVA and (+)-catechin
in AVR by HPLC-PDA analysis. Our results supported previously
published research, which indicated that ellagic acid is the major
phenolic component in the aerial parts of A. vulgaris (Møller et al.,
2009; Neagu et al., 2015; Ilić-Stojanovićet al., 2017). To the best of
our knowledge, phytochemical screening of A. vulgaris underground
parts was performed only by Geiger et al. (1994). They found condensed
tannins as major components (50% of the total tannins) of the 80%
methanolic extract of A. vulgaris roots and confirmed the presence of
ellagitannins (agrimoniin, pedunculagin, and laevigatin F) in the fresh
aerial and underground parts as well. By spectrophotometric determi-
nation, we observed a high amountof phenolic compounds in both ex-
tracts; the extracts were especially rich in condensed tannins. Maier
et al. (2017) found that the Lady's mantle herb extract contains about
30% w/w of tannins, which is in agreement with our findings. Ellagic
acid manifests a broad spectrum of biological activity (Khanduja et al.,
1999; Beserra et al., 2011). With regard to the high amount of phenolic
compounds with proven health benefits, A. vulgaris can be consideredas
a promising medicinal plant.
Free radicals and other oxidants are responsible for the emergence
of a large number of diseases, such as Parkinson's disease, cancer,
cardiovascular, and obesity-related diseases (Lobo et al., 2010). There
is increasing evidence that phenolics and other natural antioxidants
from plants in the human diet may prevent, postpone and control the
development of degenerative diseases (Consolini and Sarubbio, 2002;
Dastmalchi et al., 2012; Costa et al., 2013; Batista et al., 2014).
The results we obtained for antioxidant activity of examined extracts
through seven methods undoubtedly demonstrated the strong
antioxidant potential of extracts, comparable with reference compound
catechin and even better than synthetic preservative (BHT) in certain
antioxidant assays. AVA showed significantly better (pb0.05)
antioxidant activities in all employed methods in our study, except for
total antioxidant activity. Furthermore, AVA demonstrated particularly
better antioxidant activity in the inhibition of lipid peroxidation than
AVR. The lowest IC
50
values for the extracts were recorded in DPPH
assay. IC
50
values for the extracts were lower in comparison with
those for BHT and they are in common with the results reported by
Ilić-Stojanovićet al. (2017), while no statistically significant difference
(pb0.05) was observed between AVA and catechin. Since numerous
plant phenolics have been found to be responsible for biological proper-
ties, it can be assumed that antioxidant activities of these extracts are
Table 4
Antifungal activity of the methanolic extracts of aerial parts and rootsof A. vulgaris.
Fungal species MIC
a
values
AVA AVR Ellagic
acid
Catechin Nystatin
Phialophora fastigiata (FSB 81) 10 20 N1N1 1.25
Penicillium canescens (FSB 24) 20 20 0.25 N1 2.5
Trichoderma viride (FSB 11) N20 20 1 N1 0.078
Trichoderma longibrachiatum
(FSB 13)
N20 20 N1N1 0.078
Aspergillus brasiliensis
(ATCC 16404)
20 N20 N1N15
Aspergillus glaucus (FSB 32) 5 10 1 N15
Fusarium oxysporum (FSB 91) 10 20 1 N1 2.5
Alternaria alternata (FSB 51) 20 N20 N1N1 0.625
Doratomyces stemonitis (FSB 41) 2.5 5 N1N1 2.5
Candida albicans (ATCC 10259) N20 N20 N1N1 0.625
a
MIC values, minimum inhibitory concentrations given as mg/mL for plant extracts,
ellagic acid, and catechin, and as μg/mL for antimycotic nystatin.
180 T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
related to their phenolic profile. With respect to the high amount of
polyphenols, strong antioxidant activity under in vitro and invivo condi-
tions was reported for other species from the Rosaceae family (Katanić
et al., 2015; Jiménez-Aspee et al., 2016).
One of the main reasons to find novel natural sources of antioxidants
is the fact that a large number of reactive oxygen species is produced
during the inflammatory process (Conner and Grisham, 1996). Serious
side effects of existing anti-inflammatory drugs are burning pharma-
ceutical concern worldwide. Therefore, research goes on to find new
highly effective and harmless anti-inflammatory remedies of natural
origin which can be alternatives to NSAID’s. The undertaken study
demonstrates, for the first time, the effects of A. vulgaris methanolic
extracts on COX-1 and COX-2 enzymes inhibition, with the preferential
COX-2 inhibitory activity of AVA with AVR approximately the same
Fig. 2. COX-1and COX-2 inhibitory activities and COX-2 gene expression onTHP-1 of A. vulgarisextracts (50 and 25 μg/mL,respectively).Indomethacin,NS-398 and dexamethasone (DEX)
were used as positive controls, according to Katanićet al. (2016). Thegraph represents compiled data (% inhibition) of two independent experiments (mean ± SD).
181T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
inhibitory activity on both COX isoforms. Notwithstanding that AVR is
not COX-2 specific, the relatively high percentage of the inhibition of
COX enzymes is confirming the presence of anti-inflammatory com-
pounds in this extract. Therefore, these results can be of great impor-
tance for further testing of Lady's mantle as a potential anti-
inflammatory remedy. NF-κB is a nuclear transcription factor regulating
the expression of various genes, including IL-1β,IL-6,TNF-α, and iNOS,
which play critical roles in inflammation, apoptosis, and tumour genesis
(Lawrence et al., 2001). Negative results obtained for NF-κBproduction
in our study indicate that neither AVA nor AVR exert their anti-
inflammatory activity through the inhibition of NF-κB. Anti-
inflammatory activity of A. vulgaris has already been tested for the in-
hibition of 15-lipoxygenase activity (Trouillas et al., 2003), and the re-
sults provided a presumption that anti-inflammatory action of
A. vulgaris may be related to the inhibitory activity of phenolic com-
pounds on arachidonic acid metabolism through the lipoxygenase path-
way. According to Şeker-Karatoprak et al. (2017), methanolic and water
extracts of A. mollis decreased the nitrite as well as inhibited TNF-αpro-
duction in LPS-induced macrophages. In support of the traditional use of
Alchemilla species in wound treatment, Shrivastava et al. (2007)
displayed acceleration in wound healing and a significant reduction in
the size of dorsal skin lesions in rats by the second day of the treatment
with 3% A. vulgaris in glycerine. Also, A. vulgaris enhanced proliferation
of epithelial, liver and myofibroblasts cells as well. It is worth noting
that no cytotoxic effect or any morphological changes were observed in
the cells exposed to A. vulgaris extract in the above mentioned study.
We obtained the same results in our study through biocompatibility as-
says on immortalised fibroblasts BalbC-3 T3 and normal epidermal
HaCaT cells during 48 and 72 h. Fibroblasts and keratinocytes facilitate
the protective role of normal skin and play a crucial role in cutaneous rep-
aration process. When a wound occurs, then natural skin barrier is
disrupted, which promotes the proliferation as well as the maturation
of fibroblasts and keratinocytes. They migrate into the wound site and
communicate via certain signalling loops, which support the restoration
and regeneration of tissue homeostasis after wounding (Wojtowicz
et al., 2014). Biocompatibility of plant extracts may prevent differentia-
tion, proliferation, and attaching of these skin cells. The results of our
study indicatethat the application of AVA and AVR at doses ranging
from 10 to 50 μg/mL provides good biocompatibility, with no toxic
or injurious effects on the healthy cells.
According to Kuete (2010), an extract can be considered as a potent
antibacterial agent with significant antibacterial activity with MICs
below 0.1 mg/mL, while MICs between 0.1 and 0.625 pointed to moder-
ate activity against bacterial growth. MICs above 0.625 mg/mL referred
to weak activity. The results in Table 3 revealed that Gram-positive
bacteria are more susceptible in comparison with Gram (−) ones.
These findings are not surprising, considering the fact that Gram (−)
bacteria are more resistant than Gram (+) ones to plant extract treat-
ment, because of the porins and lipopolysaccharides present in their
outer membrane, which provides a protective barrier and prevents in-
tracellular penetration of antibiotics, especially lipophilic ones
(Apetrei et al., 2011). To the extent of our knowledge, this is the first
study covering antimicrobial activity of Alchemilla roots. Additionally,
there are no literature data related to the antifungal activity of
Alchemilla species, while only a few scientific reports provide informa-
tion about activities of the aboveground parts of Alchemilla species
against bacteria and C. albicans. Our results displayed negligible antifun-
gal activity of A. vulgaris with MICs from 2.5 to above 20 mg/mL. Our re-
sults are in accordance with the results published by Keskin et al.
(2010), indicated that ethanol extract of A. vulgaris at a concentration
of 4 mg/mL exhibited moderate antibacterial activity against ten bacte-
rial species, whereby the most sensitiveones were Gram-positive bacte-
ria, including E. faecalis.Krivokuća et al. (2015) reported that extracts of
four Alchemilla species exerted anti-Helicobacter pylori effect with MICs
Fig. 3. Effects of A. vulgarisaboveground parts (A) and roots (B)methanol extractson the viability of mouse immortalised BalbC-3 T3 fibroblasts and human normal HaCaT keratinocytes.
Dose- andtime-responsecurves of cells after 48 h (blackbars) and 72 h (greybars) incubationin the presenceof increasing concentrationsof the extracts. Cell v iability was a ssessed by the
MTT assay; the cell survival percentage was defined as described in Materials and Methods section. Values are given as means ± S.D. (n ≥3).
182 T. Boroja et al. / South African Journal of Botany 116 (2018) 175–184
ranging from 4 to 256 μg/mL. The same pattern of antibacterial activity
as for AVR and AVA has been observed for ellagic acid, where the most
sensitive strains were Gram positive bacteria M. lysodeikticus,
E. faecalis, and B. mycoides and Gram negative –A. chrococcum.Ourre-
sults showed low susceptibility of tested bacteria to catechin. Antibacte-
rial effects of A. vulgaris may be attributed to thehigh content of tannins
presented in the extracts (Djipa et al., 2000). With respect to the results
of antibacterial activity of phenolic standards, our findings indicate that
use of A. vulgaris extracts may be more beneficial than the application of
individual compounds, due to the possible synergistic effects of the
other components of the extracts.
5. Conclusion
Our results have demonstrated that methanolic extracts of aerial
partsandrootsofA. vulgaris are rich in phenolic compounds.
Through the evaluation of antioxidant, antibacterial, antifungal, and
anti-inflammatory activities, we observed the remarkable biological ac-
tivity of the tested extracts, as well as their full biocompatibility with fi-
broblasts and keratinocytes. Taking into account promising biological
activity and safety of A. vulgaris, our further research will be aimed to
investigate its biological activities under in vivo conditions. We hope
to discover the potential mechanism of biological action and to eluci-
date whether the specific biological activity is a result of the activity of
individual components or synergistic action of several constituents.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This research is financially supported by the Ministry of Education,
Science andTechnological Development of the Republic of Serbia (grant
No. 43004).
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