Food and Chemical Toxicology 111 (2018) 44–52

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Food and Chemical Toxicology

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

Mitodepressive, antioxidant, antifungal and anti-inflammatory effects of T

wild-growing Romanian native Arctium lappa L. (Asteraceae) and Veronica
persica Poiret (Plantaginaceae)
Radu Claudiu Fierascua,b, Milen I. Georgieva,c, Irina Fierascua,b,∗, Camelia Ungureanud,
Sorin Marius Avramescua,e, Alina Ortana, Mihaela Ioana Georgescua, Anca Nicoleta Sutanf,
Anca Zanfirescug, Cristina Elena Dinu-Pirvua,g, Bruno Stefan Velescug, Valentina Anutag
a
University of Agronomic Science and Veterinary Medicine, Bucharest, Romania
b
The National Institute for Research & Development in Chemistry and Petrochemistry, ICECHIM, Bucharest, Romania
c
Laboratory of Applied Biotechnologies, Institute of Microbiology, Bulgarian Academy of Sciences, Plovdiv, Bulgaria University, Romania
d
Politehnica of Bucharest, Faculty of Applied Chemistry and Material Science, Bucharest, Romania
e
Research Center for Environmental Protection and Waste Management, University of Bucharest, Bucharest, Romania
f
University of Pitesti, Department of Natural Sciences, Pitesti, Arges, Romania
g
Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

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

Keywords: The present study aims to evaluate the potential uses of hydroalcoholic extracts obtained from Romanian native
Arctium lappaL. wild-growing plants. The hydroalcoholic extracts were obtained from the burdock roots and respectively the
Veronica persicaPoiret aerial parts of birdeye speedwell. The extracts were characterised by HPLC (quantifying 13 compounds in the V.
Antioxidant persica extract, 6 compounds in the A. lappa extract and confirming the presence of arctiin and arctigenin in the
Antifungal
burdock extract). The antioxidant potential of the crude extracts was evaluated using two methods: the DPPH
Mitodepressive
Anti-inflammatory
assay (79.91% for speedwell extract, 76.23% for burdock extract) and the phosphomolybdate method
(296.5 mg/g ascorbic acid equivalents for burdock, 324.4 mg/g for speedwell). The crude extracts were found to
be active against both fungal lines used (Aspergillus niger and Penicillium hirsutum), inhibition zones - 17.1 mm
and 13.1 mm against P. hirsutum, respectively ca. 22 mm for both extracts against A. niger. The cytogenetic
effects (assessed using the Allium cepa assay) revealed a series of chromosomal aberrations and nuclear aber-
rations induced in the meristematic root cells. The anti-inflammatory effect, estimated in two inflammation
experimental models, showed a significant effect, especially for the speedwell extract. The results recommend
the evaluated extracts as promising sources of biologically-active compounds.

1. Introduction
Since ancient times, medicinal plants have been used for the treat-
ment of a tremendous array of diseases and disorders, ranging from
common colds to more serious conditions, or as antifungal or insect
repellent tools (Kristanc and Kreft, 2016). However, despite the pro-
gress made in pharmaceutical and especially drug-discovery science,
new uses of medicinal plants and new natural occurring compounds are
proposed for medical applications (Yu et al., 2017).
The two medicinal plants considered within the present study,
Arctium lappa L. (common name burdock) and Veronica persica Poiret
(commonly referred as birdeye speedwell) have a very long track record
of use in Romanian folk medicine, most often belonging to the wild-
flora, as weeds. They were selected for the present study considering
two main aspects: on one hand their common anti-inflammatory po-
tential and, on the other hand, their underuse and lack of scientific
studies on their applications. Burdock is traditionally applied for both
internal (in liver diseases, as diuretic or hypoglycaemic) or external (for
the treatment of eczema and skin infections, just to name a few)
treatment (Bojor, 2003). Therapeutic activity has been attributed to
flavonoids and lignans content of burdock roots. It has been reported
that antioxidative, anti-inflammatory and hepatoprotective effects are
due to the caffeoylquinic acid derivatives (Maruta et al., 1995; Lin
et al., 1996) and that antiproliferative and apoptotic effects are

∗
Corresponding author. University of Agronomic Science and Veterinary Medicine, Bucharest. Romania; The National Institute for Research & Development in Chemistry and
Petrochemistry, ICECHIM, Bucharest, Romania.
E-mail address: dumitriu.irina@yahoo.com (I. Fierascu).

https://doi.org/10.1016/j.fct.2017.11.008
Received 9 August 2017; Received in revised form 5 November 2017; Accepted 6 November 2017
Available online 07 November 2017
0278-6915/ © 2017 Elsevier Ltd. All rights reserved.
R.C. Fierascu et al.
attributable to the presence of arctigenin (Matsumoto et al., 2006) and
lappaol A, C and F (Ming et al., 2004).
In contrast to burdock, birdeye speedwell is a very common, short,
sprawling, annual herb. Recent studies have evaluated applications of
extracts or purified compounds from burdock roots as adjuvant antic-
ancer agents (Su et al., 2015), antitubercular (Zhao et al., 2014), anti-
inflammatory intestinal agents (de Almeida et al., 2013), neuropro-
tective (Tian et al., 2014), acne treatment (Miglani and Manchanda,
2014), as well as the anti-inflammatory potential (Vogl et al., 2013),
antibacterial, antioxidant (Stojković et al., 2013; Mocan et al., 2015),
antimutagenic and antitumor potential (Zivkovic et al., 2014) of dif-
ferent types of Veronica species. Pharmacological and biological activ-
ities of Veronica species could be attributed to their content in sec-
ondary metabolites such as iridoids, phenolic acids and flavones.
Previous studies showed that V. persica is rich in iridoids, especially
aucubin and catalpol type (Crişan et al., 2010).
The present work describes the prospective applications of burdock
roots and birdeye speedwell aerial parts hydroalcoholic extracts for
anticancer applications, treatment of different inflammatory and fungal
diseases, as well as adjuvant antioxidant agent, by studying their mi-
todepressive, anti-inflammatory, antifungal, and antioxidant activity.
Establishing the biological activities of natural products extracts is
dependent on the understanding of their in vivo intake, absorption,
metabolism and excretion. Although polyphenols have shown countless
health benefits, they have limited application as pharmaceutical pro-
ducts; even if they are in the form of glycosides and have relatively high
polarity, their limited water solubility prevents the passive in vivo ab-
sorption (Kaur and Kaur, 2014). As a result, their bioavailability is
generally low and can vary drastically among different polyphenol
classes as well as individual compounds in a particular class (Manach
et al., 2004, 2005). Besides poor permeability, many of the polyphenols
are subject to extensive gut and liver metabolism (Lewandowska et al.,
2013), limiting furthermore the therapeutic outcome of their adminis-
tration. Therefore, formulation of the resulted plant extracts in drug
delivery systems capable to ensure an adequate absorption of the active
compounds from the gastrointestinal tract is a necessary step for the in
vivo evaluation of their efficacy.
2. Materials and methods
2.1. Plant material and extraction conditions
Wild-growing Veronica persica L. and Arctium lappa L. were identi-
fied and harvested from fields in the Dobresti area, Pitesti hills
(44°57′48″N, 25°6′58″E). Multiple specimens were collected during
summer (July), paying special attention to the harvest of burdock roots
not longer than 60 cm; representative voucher specimens were de-
posited in BUAG Herbarium, Bucharest for future reference (voucher
nos. 40006 for birdeye speedwell and 40007 for burdock, respectively).
The crude extracts used for the study were obtained from ground
shade-dried plant material (20 g) in 1:1 mixture water-ethanol
(100:100 mL), as previously described (Fierascu et al., 2015, 2016), at
80 °C, for 2 h. The experiments were carried out using analytic grade
ethanol (Merck KGaA, Germany) and bidistilled water (obtained by a
GFL 2102 water still). The plant material represents the aerial part of V.
persica (flowers, leaves and stem) and roots of A. lappa. For the HPLC
analyses and for the formulation of microemulsions, the extracts were
concentrated on a Heidolph rotary evaporator system.
2.2. Chromatographic evaluation of tested extracts
The chromatographic analyses were carried out using a 1200 series
Agilent (Agilent Technologies, Darmstadt, Germany), consisting in a
binary pump (G1312B), degasser (G1379B), thermostated automated
sample processor (G1329A), column thermostat and DAD detector
(G1315B). Data acquisition and processing were performed using
45
Food and Chemical Toxicology 111 (2018) 44–52
Chemstation software (Agilent Technologies). The chromatographic
separation was performed using a Phenomenex Kinetex C18 core shell
column (150 × 4.6 mm i.d., 5 μm particle size) maintained under
constant temperature (30 °C). The mobile phase consisted in a mixture
of 0.1% trifluoracetic acid (solvent A) and acetonitrile (solvent B), at
1.0 mL/min flow rate, using the following elution program: 0 min, 3%
B; 2 min, 3% B; 15 min, 20% B; 20 min, 20% B; 35 min, 45% B; 40 min,
50% B; 40.1 min, 3% B. All solutions were filtered through a 0.45 μm
pore size filter (LABTECH VP30) and degassed by sonication. The in-
jection volume was 5 μL. The detection was set at 210 nm for the iridoid
derivatives, 270 nm for hydroxybenzoic acid derivatives, 280 nm for
flavanols, flavanones and lignans, 320 nm for the hydroxycinnamic
acids and flavones, and 370 nm for flavonols. Spectra were recorded for
each compound in the range 190–400 nm. Peak identification was
performed based on the retention times, as well as comparison with the
UV spectra of the reference compounds. Five-points calibration curves
were constructed for each of the analysed compounds (R2 > 0.999)
using commercially available reference substances. The catechins
[(+)-catechin, (+)-gallocatechin, (−)-epigallocatechin], as well as
hyperoside, quercitrin, kaempherol-3-O-glucoside and kaempherol-3-O-
rutinoside were purchased from Extrasynthese (Genay, France), while
the other reference standards (gallic acid, chlorogenic acid, caffeic acid,
p-coumaric acid, ferulic acid, quercetin, rutin, myricetin, luteolin, lu-
teolin-7-O-glucoside, apigenin, hesperetin, aucubin, catalpol) were ac-
quired from Sigma-Aldrich (St Louis, MO, USA). Since many phenolic
compounds are mostly present as glycosides in plants and a limited
number of glycoside standards were available, acidic hydrolysis of the
extracts (HCl 2N, 2 h at 85 °C, followed by ethyl acetate extraction) was
also performed for a better understanding of the relevant aglycones.
2.3. Antioxidant assays
The antioxidant activity of the crude extracts was evaluated using two
methods: the DPPH free radical scavenging method and the phospho-
molybdate method (evaluation of total antioxidant activity). The DPPH
(2,2-diphenyl-1-picrylhydrazyl) assay was performed as previously de-
scribed (Fierascu et al., 2015). The phosphomolybdate method implies
the following protocol: to a 0.3 mL sample solution were added 2.7 mL
of phosphomolybdate reagent solution (0.6 M sulfuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate). The mixtures
were incubated at 95 °C for 90 min. After cooling the samples to room
temperature, their extinction was measured at 695 nm on a Unicam
Helios α Thermo Orion UV-VIS spectrophotometer. Antioxidant capa-
city is expressed in ascorbic acid equivalent to 1 mg of active substance.
The calibration curve for ascorbic acid is linear in the range of
0.001–1 mg/mL, n = 5, R2 > 0.994 (Pandey et al., 2016). All ex-
periments were carried out in triplicate.
2.4. Determination of antifungal effect
The antifungal activity of the crude extracts was tested against two
relevant fungal strains Aspergillus niger ATCC 15475 and Penicillium
hirsutum ATCC 52323 by Kirby-Bauer antibiotic testing (KB testing or
disk diffusion antibiotic sensitivity testing) (Bauer et al., 1966;
Ungureanu et al., 2016).
The moulds were grown on potato-dextrose agar (abbreviated
“PDA”) from Sigma-Aldrich with the following composition: potato
extract, 4 g/L., Dextrose, 20 g/L, agar, 15 g/L. The inhibition zone
evaluation was performed according to National Committee for Clinical
Laboratory Standards (NCCLS) standards (NCCLS, 2006; Barbinta-
Patrascu et al., 2014). The stock culture was kept at 4 °C. Briefly, 20 mL
of sterile medium were poured into the Petri dish and the micro-
organism strain (1 mL) was spread on agar plates; using a sterile
Durham tube (6 mm diameter), the wells were prepared according to
the number of samples. The wells were inoculated with 50 μL sample.
All the test plates were incubated at 37 °C for 144 h, in order to allow
R.C. Fierascu et al.
microbial growth. Antifungal behaviour was estimated by observing the
ability of the sample to stop the moulds proliferation around it (i.e. the
inhibition halo). In this study triplicate plates were prepared for each
sample. Data are expressed as mean standard deviations of a re-
presentative three similar experiments carried out in triplicate. Statis-
tical analysis was performed with Origin software. Student's t-test was
used to determine the significant differences among the groups, and p-
values less than 0.05 were considered statistically significant.
2.5. Allium test
The cytogenetic effects of A. lappa and V. persica extracts were
evaluated by monitoring the changes in mitotic index (MI), chromatin
aberrations and cell abnormalities frequencies in root tips cells of Allium
cepa L., the method being previously described (Sutan et al., 2016).
Small bulbs (3–4 cm in diameter) of common onion (Allium cepa,
2n = 16) were used for the assay. For each sample, three bulbs were
placed in tap water for 48 h and transferred afterwards in tubes con-
taining hydroalcoholic extracts 5%, 25%, 50% and 100%, respectively,
for 24 h. The working concentrations were obtained by diluting the
crude extracts with the hydroalcoholic extraction solvent. Tap water
and solvent (water: ethanol = 1:1) served as controls. Roots with sui-
table lengths (1–1.5 cm) were removed from the bulbs, fixed in 3:1 (v/
v) ethanol: glacial acetic acid and stored overnight at 4 °C; the next day
they were transferred in 70% (v/v) aqueous alcohol and refrigerated at
4 °C until used. Five root tips for each plant species and each con-
centration tested, respectively, were hydrolysed in 1 N hydrochloric
acid (HCl) for 14 min. For providing a good colour contrast, root tips
immersed in hydroalcoholic extracts of A. lappa were stained for
14 min, while root tips treated with hydroalcoholic extracts of V. persica
were stained for 20 min in 2% (w/v) acetic orcein. Five slides were
made for each bulb. Each slide was examined using an Olympus CX-31
microscope and photomicrographs of cells were taken at 40 × magnifi-
cation.
For the determination of cytogenetic effects, the following para-
meters were used: (i) the mitotic index (MI); (ii) chromatin aberrations;
(iii) binucleated cells and cell abnormalities. MI was computed by de-
termining the number of cells which undergone mitosis divided by total
cells in a microscopic field into 100. Binucleated cells were scored from
the same number of interphase cells. Results are expressed in percen-
tage as mean ± standard error of several independent experiments.
The data were analysed for statistical significance using analysis of
variance (one-way ANOVA) and Duncan test was used to determine
significant differences among means. Significant differences were set at
P ≤ 0.05.
2.6. Anti-inflammatory assay
2.6.1. Extracts formulation
Since the crude hydroalcoholic extracts could not be administered
as such in the in vivo experiments (because of the high quantity of al-
cohol of the formulation) and concentration of the extract and its ad-
ministration as suspension would have led to very low absorption of the
active compounds (Lewandowska et al., 2013; Thilakarathna and
Rupasinghe, 2013), an alternative technological approach was selected.
In order to ensure appropriate bioavailability of the active compounds
from the dried extracts, formulation as microemulsion was selected as
technological approach, since many studies emphasize the numerous
advantages in terms of polyphenols in vivo absorption and metabolism
(Cui et al., 2005; Li et al., 2011).
The crude extracts were concentrated at 40 °C under vacuum using a
rotary evaporator and then lyophilized at −55 °C (CoolSafe ScanVac
55; LaboGene, Denmark). The dried extracts were stored in amber glass
recipients, at 4 °C until use.
The extracts were formulated as microemulsions and tested for anti-
inflammatory action (Rios-Hoyo et al., 2014; Mahmood et al., 2015). In
46
Food and Chemical Toxicology 111 (2018) 44–52
order to obtain the appropriate composition of the microemulsion
systems, several experiments were carried out, establishing the ade-
quate oil, surfactant, and cosurfactant constituents of the microemul-
sion. Considering the results of the preliminary experiments, oleic acid
was used as the oil phase, Tween 80 was used as the surfactant, and
propylene glycol (PG) as cosurfactant. The behaviour of the micro-
emulsions was studied by pseudo-ternary phase diagrams, using the
water titration method. The ratio of surfactant to co-surfactant was
fixed at 1:1. Oleic acid was mixed with the surfactant: co-surfactant ad
different ratios (1:9, 2:8, 3:7, 4:6, 5:5. 6:4, 7:3, 8:2, 9:1) and distilled
water was added to the mixture, in 100 μL increments. After 24 h, the
samples were evaluated in terms of homogeneity and transparency,
those which remained homogeneous and transparent, being regarded as
microemulsions. The microemulsions containing A. lappa and V. persica
extracts were obtained using the same procedure, by dispersing the
extract into the surfactant: cosurfactant mixture.
For the study of the microemulsion formulations, conductibility
studies were performed using a Corning 441 conductivity meter
(Corning, NY, USA). The refractive index was determined at 25 °C using
a digital Abbe refractometer. The mean diameter of the droplets and the
Zeta potential of the microemulsion were measured using a Mastersizer
2000 (Malvern, UK) particle size analyser.
2.6.2. Study design
68 male Wistar rats (212 ± 45 g) from the University of Medicine
and Pharmacy, Bucharest animal facility (rodent farm) were used for
the experiments. The rats were housed in plastic cages in air-condi-
tioned room and fed with granulated food with free access to water.
Temperature and relative humidity were continuously monitored with
a thermohygrometer (temperature 20–22 °C, relative humidity -
35–45%).
The study involving animal experiments was conducted in ac-
cordance with the European Community guidelines (2010/63/EU) and
had the approval of the local ethics committee (“Carol Davila” Medicine
and Pharmacy University, Faculty of Pharmacy, Bioethic Inflammation)
was evaluated in two inflammation experimental models by plethys-
mometry (Ugo Basile 7140 Plethysmometer), as previously reported
(Badea et al., 2014).
Inflammation was induced by intraplantar administration of 0.2 mL
inflammatory agent (0.6% solution of dextran and respectively 10%
aqueous suspension of kaolin) into the rat's inferior right paw. The anti-
inflammatory effect was compared with a negative control group (un-
treated rats), a positive control group (rats treated with the micro-
emulsions vehicle) and a reference substance (diclofenac).
The animals were randomly distributed in groups of 8; in both sets
of experiments (dextran and kaolin inflammatory agents), 1 mL/100 g
body weight (b.w.) distilled water was administered the negative con-
trol group; the positive control group animals were treated with the
microemulsions vehicle, 1 mL/100 g b.w., the reference group animals
were treated with 1% sodium diclofenac solution, 100 mg/kg b.w.,
while the two experimental groups were treated with 1 mL/100 g b.w.
of microemulsions containing speedwell extract (A) and burdock ex-
tract (B), respectively. After the administration of substances by gavage,
all animals were intraperitoneal anesthetized with 13% solution of
urethane 1.3 g/kg b.w. and the initial paw volume was measured (V0).
The paw oedema was further evaluated at 1, 2, 3, 4, 5 and 24 h re-
spectively, after the inflammatory agent administration.
The statistical analyses were performed using GraphPad Prism
version 5.00 for Windows, GraphPad Software, San Diego California
USA. The evolution of paw oedema was calculated using the formula:
Vxh − V0
%= × 100
V0 (1)
where V0 is the initial paw volume and Vxh is the paw volume corre-
sponding to the time measurement. The anti-inflammatory effect was
calculated as the difference between the evolutions of paw oedema of
R.C. Fierascu et al. Food and Chemical Toxicology 111 (2018) 44–52

Table 1 quantified glycosides, luteolin-7-O-glucoside and rutin were present in

Quantification of the selected compounds found in the V. persica and, respectively A. significant amounts (approximately 330 μg/g d.w.).
lappa extracts; the concentrations are expressed in μg/g d.w. plant material; the con-
The acid hydrolysis of the extract provided useful information with
centrations of the aglycones obtained after acid hydrolysis of the extracts are presented in
parenthesis. respect to the relevant aglycones for V. persica: three major peaks were
obtained, corresponding to luteolin, apigenin and quercetin (results are
Compound Rt (min) λ (nm) Calibration Concentration (μg/g presented in Table 1).
range (μg/ d.w.) The burdock extract is dominated by phenolic acids. The major peak
mL)
V. persica A lappa
in the chromatogram corresponds to chlorogenic acid (4.388 mg/g
d.w.). The other major peaks (Rt = 19.42 and Rt = 21.65 min) were
Gallic acid 3.69 270 0.25–100 3.904 19.069 identified as arctiin and its aglycone, arctigenin, based on the matching
Chlorogenic acid 11.24 320 0.25–100 121.622 4388.288 of their spectra with the spectra of the reference compounds from our
Caffeic acid 11.88 320 0.25–100 218.469 25.676
p-coumaric acid 14.99 320 0.25–100 – 7.958
database. However, since reference standards were not available at the
Ferulic acid 16.33 320 0.25–100 372.973 – time of the present study, the results were limited to confirming their
(+)-catechin 12.84 280 0.4–80 – – presence in the extract. All other signals from the burdock chromato-
(−)-epigallocatechin 10.74 280 0.2–80 – 1447.447 gram presented phenolic acid-type UV spectra.
(−)-epicatechin 13.49 280 0.4–80 – 496.397
Quercetin 26.21 370 0.25–100 25.075 –
(528.829) 3.2. Antioxidant and antifungal effect
Quercitrin 18.22 370 0.5–100 1.802 –
Rutin 16.62 370 0.5–100 332.733 – The antioxidant activity of the crude extracts was evaluated fol-
Hyperoside 19.40 370 0.5–100 160.661 – lowing the DPPH radical scavenging assay. The antioxidant potential of
Kaempferol-3-O- 17.01 370 0.5–100 – –
glucoside
the crude extracts was found to be 79.91 ± 0.31% for V. persica extract
Kaempferol-3-O- 19.09 370 0.5–100 15.165 – and, respectively, 76.23 ± 0.29% for A. lappa extract. The total anti-
rutinoside oxidant capacity of the two extracts followed the same trend
Luteolin 26.67 320 0.2–20 38.589 – (324.4 mg/g ascorbic acid equivalent and, respectively, 296.5 mg/g
(632.583)
ascorbic acid equivalent). The higher antioxidant effect of the speed-
Luteolin-7-O- 17.22 320 0.35–35.2 329.279 –
glucoside well extract may be linked to the higher content of polyphenols or to
Myricetin 20.87 370 0.35–35.2 – – their iridoid content (Hanganu et al., 2016).
Apigenin 24.41 320 0.2–20 51.051 – The antifungal activity of the two extracts was determined using
(295.946) miconazole nitrate and solvent (ethanol: H2O = 1:1) as positive and
Hesperetin 29.86 280 0.25–100 – –
negative control, respectively. Fig. 2 presents the diameters of inhibi-
Arctiin 19.48 280 NA – Present
Arctigenin 21.65 280 NA – Present tion zones (in millimetres) against Aspergillus niger ATCC 15475 and
Catalpol 2.29 210 0.8–80 127.027 – Penicillium hirsutum ATCC 52323.
Aucubin 2.82 210 0.8–80 769.520 – Both studied extracts present antimicrobial effect, burdock extract
being slightly more efficient on P. hirsutum than the speedwell extract
(inhibition zones - 13.1 mm and 17.1 mm, respectively); both extracts
the treated groups and the negative control group or the reference
show a similar effect on A. niger (ca. 55% of the positive control in-
group. Results are expressed as mean ± standard deviation. The ap-
hibition zone diameter). The results are in agreement with literature
plied parametrical tests (t-test, one-way ANOVA) have a 90% con-
data: thus, Stojković et al. (2013), studying Veronica montana L. water
fidence interval and statistical differences were considered for a p
extract, suggested antimicrobial effect of the extract against six strains
value < 0.05.
of Gram-positive and Gram-negative bacteria, while Mocan et al.
(2015), investigating V. officinalis, V. teucrium and V. orchidea, obtained
3. Results and discussions MIC values for Staphylococcus aureus, Listeria monocytogenes and Listeria
ivanovii ranging between 3.9 and 15.62 mg/mL. Habibipour and Rajabi
3.1. Analytical characterisation (2015) examined the antibacterial effect of Arctium lappa hydroalco-
holic extract on several microorganisms, including Pseudomonads aer-
The results obtained by the HPLC analysis of the extracts are pre- aginosa, Haemophilus influenza, Bacillus subtilis, Bacillus cereus, Klebsiella
sented in Table 1. pneumonia and Staphylococcus aureus, suggesting the potential of A.
The typical chromatograms obtained for V. persica and A. lappa lappa extract for replacing synthetic antimicrobial drugs. Lou et al.
extracts, as well as the chromatogram of a standard sample containing (2016), studying the burdock leaf efficiency in meat preservation, at-
18 of the studied compounds are presented in Fig. 1. Since the iridoid tributed the antimicrobial properties to the phenolic acids content (such
derivatives (aucubin, catalpol) can only be detected at λ < 210 nm, as chlorogenic acid, rutin, quercitrin, luteolin, p-coumaric acid, caffeic
for the V. persica extract the chromatogram is presented as the overlaid acid and quercetin).
signal of the 5 monitored wavelengths, whereas for the rest of the Numerous studies have suggested that the defence of some plants
compounds, only the signals corresponding to λ = 280 nm are pre- against fungal pathogen is probably one of the primary function of
sented. secondary metabolites in plants (Cazetta et al., 2008). The studied ex-
The major iridoid compound in V. persica extract was found to be tracts contain two important classes of secondary compounds, iridoids
aucubin (769.520 μg/g d.w.), with a content of approximately 6 times and phenolics; iridoids have numerous biological activities, including
higher than catalpol. A major unidentified peak eluted at 14,85 min, its antifungal and antioxidants effects (Graikou et al., 2002), while the
spectrum being an indicative of a flavone glycoside (λmax = 334 nm). antifungal and antioxidant activity of phenolic compounds has been
The other major peak (Rt = 21.34 min), was identified as o-para- previously reported (Cafarchia et al., 1999; Lou et al., 2016). It can be
hydroxybenzoic acid, by matching the spectrum versus our spectral concluded that the antifungal activity is mainly due to the phenolic
database. Based on the same method, the peak at Rt = 9.18 min was compounds present in the studied extracts.
identified as protocatechuic acid. Most of the unidentified peaks in the
15–23 min range presented spectra corresponding to flavonoid glyco- 3.3. Cytogenotoxicity evaluation
sides, whereas the peaks with retention times of less than 14 min had
spectral characteristics consistent to phenolic acids. Among the The microscopic results of A. cepa L. roots exposed to 4 different

47
R.C. Fierascu et al. Food and Chemical Toxicology 111 (2018) 44–52

Fig. 1. HPLC chromatograms of the used standards (A) and the two extracts: speedwell (B) and burdock (C).

concentration of V. persica and A. lappa hydroalcoholic extracts are A. lappa extract compared with the 50% A. lappa extract that presented
summarized in Figs. 3–5. MI significantly decreased under the influence the highest division rate. Otherwise, the treatment of V. persica extract
of both extracts, compared with the negative control, after 24 h of seems to have a slighter mitodepressive effect, since 5%, 25% and 100%
treatment (Fig. 3). Stronger mitodepressive effect was obtained for 5% variants show significantly (p < 0.05) elevated effects in meristematic

48
R.C. Fierascu et al.
Fig. 2. Inhibition zone diameters measured for negative control, V. persica extract, A.
lappa extract and positive control against the tested fungal lines Aspergillus niger (red) and
Penicillium hirsutum (blue), including statistical interpretation; Values presented re-
presents means ± standard deviation; means in a series without a common superscript
letter differ (P < 0.05) as analysed by one-way ANOVA and the Tukey test. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Fig. 3. Effect of Arctium lappa L. and Veronica persica Poiret hydroethanolic extracts on
mitotic index of Allium cepa L. root tip cells (a, b, c represents the interpretation of the
significance of the differences by means of the Duncan test, p < 0.05).
Fig. 4. Effect of Arctium lappa L. and Veronica persica Poiret hydroethanolic extracts on
frequency of binucleate cells in Allium cepa L. root tips (a-f represents the interpretation of
the significance of the differences by means of the Duncan test, p < 0.05).
roots cells of A. cepa. For both extracts, the mitotic index varied in-
dependently of the tested concentration. Mitodepressive effect may be
the result of the blockage in phase G1 of the cell cycle (El-Ghamery
et al., 2000) or of the suppressive effect of some components on DNA
and nucleoprotein synthesis (Schulze and Kirscher, 1996). Quercetin,
the most abundant flavonoids, may be responsible for the inhibition of
49
Food and Chemical Toxicology 111 (2018) 44–52
cell cycle progression, acting as a disruptor of microtubules poly-
merization or as an inhibitor of kinases activity (Jackson and Venema,
2006; Boly et al., 2011). Quercetin-3-O-glucoside could also be in-
criminated for mitotic inhibition. Sudan and Rupasinghe (2015) proved
that quercetin-3-O-glucoside cause alteration hepatocellular carcinoma
cells cycle progression via induction of S-phase arrest. Our results
suggest that V. persica and A. lappa extracts have an antiproliferative
potential eventually. In agreement with these results, previous studies
showed selective antiproliferative in vitro activity of A. lappa L. root
extracts in human cancer cell lines (Predes et al., 2011) and potential
anticancer therapeutic effect of lappaol F extracted from A. lappa seeds
(Sun et al., 2014).
No aberration was observed in the A. cepa exposed to control.
Chromatin aberrations and nuclear abnormalities frequencies in root
tips cells of A. cepa L. treated with A. lappa extract were apparently
different from that caused by V. persica extract. According to micro-
scopic results, starting from 50% concentration, A. lappa extract in-
duced C-mitosis and abnormal anaphases (Fig. 4) with a very low fre-
quency (2.28% in average), and an important percentage of cells in
interphase were binucleate. A concentration-related decrease in the
frequency of binucleate cells was produced by both extracts (Fig. 3).
Binucleate cells represent the marker of nuclear abnormalities in-
duced by the tested extracts, since they were omnipresent, regardless
the type of extract or the concentration tested. This result strengthens
the observation that burdock and birdeye speedwell hydroethanolic
extracts inhibit mitosis. Relatively high frequency of binucleate cell
represents the consequence of spindle aberrations at early anaphase
(Roper, 1977) or inhibition of cytokinesis following telophase (Nefic
et al., 2013). Another hypothesis for the alteration of cell division, and
cytokinesis respectively, could be the inhibition of cell-plate formation
or dispersion of cell-plate after formation, due to some chemicals that
disrupts the Golgi apparatus and block the supply of vesicles carrying
cell-plate materials (Nishihama and Machida, 2001). The study of
Salmela et al. (2009) showed that dietary flavonoid fisetin has the
ability to act as inhibitor of cell proliferation that induce failure of
cytokinesis. Other phenolic compounds such as gallic acid or ferulic
acid have been previously tested for their cytotoxicity and antic-
arcinogenic activity (Kumar and Pruthi, 2014). A maximum of 2 bi-
mitosis (Fig. 4) were observed per 3000 cells, which is in accordance
with the previous data showing the mitodepressive effect of the tested
extracts. Very rarely, the presence of apoptotic bodies and altered cells
were also noticed, like gigantic and extended cells (Fig. 5) in A. cepa
root tip cells exposed to V. persica hydroethanolic extracts for 24 h. This
lower toxicity of speedwell could be explained by the higher total
phenolics content.
Although the in vitro cytotoxicity assay cannot be directly related to
the in vivo toxicity, the results of our study have an important con-
tribution to a better understanding of the antitumor potential of the
tested extracts.
It is worth to be noticed that root tips of A. cepa incubated in V.
persica extracts required an increased time for orcein staining, in order
to obtain the favourable contrast. Orcein dye is very suitable for so-
matic chromosomes in plants due to its capacity of staining both DNA
and protein. A weak staining could be the consequence of protein re-
moval or inactivation of tertiary amines of the chromosomal polypep-
tide (Sharma and Sen, 2002). Using the Taenzer-Unna acid orcein
method, Fullmer and Lillie (1956) suggested that polar groups such as
OH, NH2 and COOH might interfere with orcein reactivity. In our study,
weak staining may be due to improper fixation and clarification of
chromosome morphology, followed by interaction with OH groups of
phenols.
3.4. Anti-inflammatory assay
As already mentioned, the microemulsions with the highest stability
and lower mean droplet size have been selected for use in experiments.
R.C. Fierascu et al. Food and Chemical Toxicology 111 (2018) 44–52

Fig. 5. Chromosomal aberrations and nuclear aberrations identified

in meristematic root cells of A. cepa L. that underwent treatment
with A. lappa and V. persica hydroalcoholic extracts: (a) binucleate
cells - 25% extract of A. lappa; (b, c) bimitosis - 50% A. lappa; (d) C-
mitosis and aberrant anaphase - 100% extract of A. lappa; (e)
apoptotic bodies – 50% extract of V. persica; (f) weak stained of
metaphase and anaphase – 25% extract of V. persica.

The general selected composition consisted in 5% dry plant extract,
9.29% oleic acid, 57.14% Tween 80: PG 1:1 (w/w) mixture and 28.57%
water, which ensured formation of stable oil in water microemulsions
with mean droplet size of 92 ± 6 nm for the empty microemulsions
and 117 ± 7 nm for the extract loaded ones. The recorded Zeta po-
tential was – 39.3 ± 3.7 mV for the empty systems, and
−21.1 ± 3.8 mV for the loaded ones, also indicative of stable for-
mulations.
The mechanisms of bioavailability enhancement due to the micro-
emulsion formulation are multiple, and include: keeping the active
compounds from the extract in solution and avoiding therefore the
initial dissolution step (Pouton, 1997), stimulation of biliary secretions
associated with micellar solubilization of poorly soluble compounds,
stimulation of the lymphatic transport, prolongation of the gastro-
intestinal residence time and reduced metabolism and efflux pump
activity (Narang et al., 2007). The surfactants and cosurfactants from
the formulation reversibly increase polyphenols permeability, by dis-
rupting the lipid bilayer of the epithelial cell membrane (Gursoy and
Benita, 2004). The microemulsions are also capable to alter the main
absorption site of most flavonoids, from the colon to the small intestine,
with higher absorption rates and lower variability (Walle, 2004).
The anti-inflammatory effect of the extract microemulsions was
evaluated in two acute inflammation experimental models by plethys-
mometry method, compared to two control groups (untreated rats and
rats treated with the microemulsion vehicle) and a reference substance
(diclofenac).
The results of the plethysmometric measurements were statistically
analysed in order to obtain paw oedema evolution for the groups with
dextran and kaolin induced inflammation respectively. These data are
summarized in Tables 2 and 3.
For the dextran induced inflammation model, the paw oedema vo-
lume was significantly higher compared to the initial values (t-Test,
p < 0.05) for all the animal groups, with no return to baseline at the
end of the observation period, with an exception for the animals treated
with microemulsion (A). The obtained results indicate that the micro-
emulsion containing V. persica extract reduces the dextran induced paw
oedema of the rats at the end of the observation period to values similar
to V0.
Fig. 6 presents the mean paw oedema evolution for the dextran
inflammatory model.
For the dextran induced inflammation model, the global paw oe-
dema evolution process for the groups treated with microemulsions (A)
and (B) was different when compared to the negative control group and
to the group treated with diclofenac (ANOVA, p < 0.05). The micro-
emulsion vehicle influenced the global paw oedema evolution process
(ANOVA, p < 0.05), but the process was similar to the negative con-
trol group at the measurement times (t-test, p > 0.05).
The microemulsion B (containing burdock) showed a similar paw
oedema evolution with the group treated with diclofenac (t-test,
p > 0.05), at all the measurement times, microemulsion A (containing
speedwell extract) had a similar paw oedema evolution after 1 and 2 h
with the group treated with diclofenac (t-test, p > 0.05). Animals
treated with the microemulsion (A) showed a maximum anti-in-
flammatory effect at the end of the experiment.
For the kaolin induced inflammation model the paw oedema vo-
lume was significantly higher compared to the initial values
(p < 0.05) for all the animal groups, without return to baseline at the
end of the observation period.

Table 2
The results of the plethysmometric measurement of the paw oedema induced with dextran and statistical interpretation.

Experimental group V0 V1h V2h V3h V4h V5h V24h

Negative 1.19 ± 0.07 2.02 ± 0.15 2.01 ± 0.14 2.15 ± 0.17 2.11 ± 0.15 2.07 ± 0.10 1.43 ± 0.10
control t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Positive control 1.11 ± 0.08 1.76 ± 0.23 1.91 ± 0.17 1.83 ± 0.14 1.85 ± 0.19 1.89 ± 0.15 1.32 ± 0.11
t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Treated with 1.13 ± 0.09 1.71 ± 0.18 1.70 ± 0.16 1.79 ± 0.14 1.86 ± 0.15 1.72 ± 0.19 1.40 ± 0.22
diclofenac t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Treated with 1.02 ± 0.08 1.75 ± 0.19 1.77 ± 0.21 1.76 ± 0.22 1.70 ± 0.23 1.64 ± 0.19 1.04 ± 0.07
(A) t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 > 0.05
Treated with 1.10 ± 0.05 1.73 ± 0.11 1.77 ± 0.20 1.71 ± 0.13 1.86 ± 0.17 1.70 ± 0.17 1.18 ± 0.11
(B) t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05

Where V, the volume of oedema; t-Test, p.

50
R.C. Fierascu et al. Food and Chemical Toxicology 111 (2018) 44–52

Table 3
The results of the plethysmometric measurement of the paw oedema induced with kaolin and statistical interpretation.

Experimental group V0 V1h V2h V3h V4h V5h V24h

Negative control 1.18 ± 0.08 1.86 ± 0.10 1.89 ± 0.12 1.95 ± 0.11 2.01 ± 0.11 2.02 ± 0.07 1.96 ± 0.12
t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Positive control 1.03 ± 0.08 1.36 ± 0.08 1.53 ± 0.14 1.54 ± 0.14 1.58 ± 0.15 1.42 ± 0.12 1.41 ± 0.12
t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Treated with 1.14 ± 0.07 1.71 ± 0.17 1.82 ± 0.18 1.83 ± 0.13 1.90 ± 0.24 1.86 ± 0.11 1.96 ± 0.18
diclofenac t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Treated with 1.00 ± 0.07 1.41 ± 0.13 1.48 ± 0.16 1.53 ± 0.11 1.58 ± 0.15 1.59 ± 0.16 1.58 ± 0.19
(A) t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05
Treated with 1.02 ± 0.07 1.46 ± 0.13 1.56 ± 0.18 1.58 ± 0.14 1.61 ± 0.22 1.58 ± 0.12 1.82 ± 0.24
(B) t-Test,p < 0.05 < 0.05 < 0.05 < 0.05 < 0.05 < 0.05

Where V, the volume of oedema; t-Test, p.

previously studied for their free radical scavenging or antioxidant ac-

tivity, polyphenols being responsible for the antioxidant activity of
several natural extracts (Khatun et al., 2017). Literature data links the
antioxidant activity of the polyphenols with their redox properties
(Khatun et al., 2017; Nitin et al., 2010). Also, several studies presented
other extracts rich in polyphenols with biological activities (cytotoxic,
anticancer, antibacterial, just to name a few) (Khatun et al., 2017). The
superior antioxidant and anti-inflammatory effects observed for the
speedwell extract can probably be attributed to its content in iridoid
compounds and polyphenols (Dussossoy et al., 2011).

4. Conclusions

Fig. 6. Mean paw oedema evolution for the dextran-induced inflammation. The changes noticed in the cells demonstrate that A. lappa and V.
persica hydroethanolic extracts exhibit mitodepressive effects in mer-
istematic root cells of A. cepa, suggesting their antiproliferative activity.
The microemulsions containing the speedwell and burdock extracts
exerted anti-inflammatory effects in the performed experimental
models. The microemulsion vehicle did not interfere with the extracts
effect. The higher effect obtained after the administration of speedwell
extract as microemulsions may recommend this formulation for further
studies as dietary factors in pathologies with inflammatory component.

Acknowledgements

The authors gratefully acknowledge the support obtained through

the project SusMAPWaste, SMIS 104323, Contract no. 89/09.09.2016,
from the Operational Program Competitiveness 2014–2020, project co-
Fig. 7. Mean paw oedema evolution for the kaolin inflammatory model. financed from the European Regional Development Fund.

In Fig. 7 is presented the mean paw oedema evolution for the kaolin
inflammatory model.
For the studied inflammation model, the global paw oedema evo-
lution process for the groups treated with microemulsions (A) and (B)
was different compared to the negative control group and to the group
treated with diclofenac (ANOVA, p < 0.05). The microemulsion ve-
hicle did not influence the global paw oedema evolution process, the
process being similar to the negative control group (ANOVA,
p > 0.05). The microemulsions (A) and (B) have a similar paw oedema
evolution after 5 and 24 h compared to the group treated with diclo-
fenac (t-test, p > 0.05).
Literature data suggest that the polyphenols contained by the stu-
died hydroalcoholic extracts may interfere with the increase of the
vascular permeability generated by the release of histamine and ser-
otine caused by dextran and the increase of the proinflammatory cy-
tokines caused by kaolin (Jajtner et al., 2016; Bezerra et al., 2017; Guo
et al., 2017; Zeinali et al., 2017). The anti-inflammatory effect exerted
by the microemulsions containing the extracts may be linked to the
increase of the polyphenols bioavailability.
Plant constituents (polyphenol compounds, for example) were
Transparency document
Transparency document related to this article can be found online at
http://dx.doi.org/10.1016/j.fct.2017.11.008.
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