Received: 7 June 2018 Revised: 12 July 2018 Accepted: 13 July 2018

DOI: 10.1002/ptr.6174

RESEARCH ARTICLE

Artichoke leaf extract, as AKR1B1 inhibitor, decreases sorbitol

level in the rat eye lenses under high glucose conditions ex vivo
Ivana Šušaníková1 | Jana Balleková2 | Milan Štefek2 | Jan Hošek3 | Pavel Mučaji1

1
Department of Pharmacognosy and Botany,
Faculty of Pharmacy, Comenius University, In the previous study, the artichoke leaf extract showed effective inhibition of AKR1B1,
Bratislava, Slovakia
the first enzyme of polyol pathway, which reduces high level of glucose to osmotically
2
Institute of Experimental Pharmacology and
Toxicology, Slovak Academy of Sciences, active sorbitol, important for development of chronic diabetic complications. In the
Bratislava, Slovakia present study, the effect of artichoke leaf extract and of several participating phenols
3
Department of Molecular Biology and
(caffeic acid, chlorogenic acid, quinic acid, and luteolin) was tested on sorbitol level in
Pharmaceutical Biotechnology, Faculty of
Pharmacy, University of Veterinary and rat lenses exposed to high glucose ex vivo, on cytotoxicity as well as on oxidative stress
Pharmaceutical Sciences Brno, Brno, Czech
in C2C12 muscle cell line induced by high glucose in vitro. The concentration of sorbitol
Republic
Correspondence
was determined by enzymatic analysis, the cytotoxicity was provided by WST‐1 test
Ivana Šušaníková, Department of and intracellular content of reactive oxygen species was determined by fluorescence
Pharmacognosy and Botany, Faculty of
Pharmacy, Comenius University in Bratislava,
of 2′‐7′‐dichlorofluorescein probe. The extract and the compounds tested showed
Odbojárov 10, 83232 Bratislava, Slovakia. significant protection against toxic effects of high concentration of glucose in both
Email: ivana.susanikova@gmail.com
models. On balance, the artichoke leaf extract thus represents a prospective preventive
Funding information
Agentúra Ministerstva školstva, vedy, agent of development of chronic diabetic complications, probably due to phenols
výskumu a športu SR, Grant/Award Numbers: content, concerning preclinical and clinical studies.
APVV‐15‐0308 and APVV‐16‐0207; Faculty
of Pharmacy, Comenius University in Brati-
slava, Grant/Award Number: FaF UK/6/2016; KEY W ORDS
Ministerstvo školstva, vedy, výskumu a športu aldo‐keto reductases, polyol pathway, oxidative stress, chronic diabetic complications, Cynara
Slovenskej republiky, Grant/Award Numbers:
cardunculus, glucose toxicity
VEGA 1/0290/16, VEGA 1/0359/18, VEGA
1/0561/18 and VEGA 2/0041/15; Veterinární
a Farmaceutická Univerzita Brno, Grant/
Award Number: IGA VFU 313/2018/FaF

1 | I N T RO D U CT I O N Chlorogenic, caffeic, and quinic acids, present in the extract

Our previous study reported on biological activities of artichoke leaf
extract (Miláčková, Kapustová, Mučaji, & Hošek, 2017). The extract
effectively inhibited isolated human aldose reductase AKR1B1
(IC50 = 5.12 ± 0.11 μg/ml) and its rat equivalent rat lens aldose reduc-
tase (RL‐AKR; IC50 = 3.06 ± 0.08 μg/ml) and effectively reduced
NF‐κB activity during LPS incubation in human leukemic monocytes.
Abbreviations: AKR1B1, human aldose reductase; Art, artichoke leaf extract;
CA, caffeic acid; ChA, chlorogenic acid; COX‐2, cyclooxygenase‐2; DCF, 2′‐7′‐
dichlorofluorescein; DCFH‐DA, 2,7‐dichlorodihydrofluorescein diacetate;
DMEM, Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; LPS,
bacterial lipopolysaccharide; Lut, luteolin; MMP‐2, matrix metalloproteinase
type 2; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide
adenine dinucleotide phosphate; PBS, phosphate buffered saline; QA, quinic
acid; RL‐AKR, rat lens aldose reductase; ROS, reactive oxygen species; SDH,
sorbitol dehydrogenase
(Kuczmannová et al., 2015), may be responsible for these effects.
The aldo‐keto reductase superfamily includes more than 150 structur-
ally related NAD(P)H‐dependent enzymes, which reduce carbonyls to
their appropriate alcohols. AKR1B1, a member of this family, is
included in the intracellular inflammatory pathway as well as the first
enzyme of the polyol pathway, in which it reduces glucose to osmotic
active alcohol sorbitol under hyperglycemic conditions. The sorbitol
accumulation in tissues may result in disruption of cellular osmotic
homeostasis (Kador, Lee, Fijisawa, Blessing, & Lou, 2000). In addition,
the increased flux of glucose through the polyol pathway and conse-
quent depletion of nicotinamide adenine dinucleotide phosphate
(NADPH) may inhibit the activity of other NADPH‐requiring enzymes,
including those of the glutathione redox cycle. The decreased levels of
reduced glutathione increase the susceptibility of cells to damage by
oxidative stress (Hamada, Araki, Horiuchi, & Hotta, 1996; Obrosova,

Phytotherapy Research. 2018;1–7. wileyonlinelibrary.com/journal/ptr © 2018 John Wiley & Sons, Ltd. 1
2 ŠUŠANÍKOVÁ
2005). This mechanism, along with other factors of glucose toxicity
like protein‐kinase C‐dependent activation of NAD(P)H oxidase, could
lead to increase of oxidative stress. Osmotic stress together with oxi-
dative stress may contribute to the development of chronic diabetic
complications as cataract, diabetic neuropathy, nephropathy, and
others (Alexiou, Pegklidou, Chatzopoulou, Nicolaou, & Demopoulos,
2009; Kador, 1998; Yabe‐Nishimura, 1998). Antioxidants and aldose
reductase inhibitors were recognized as important factors in the strat-
egy of prevention and attenuation of long‐term diabetic complications
(Coudert et al., 1994; Juranek, Horakova, Rackova, & Stefek, 2009;
Karasu et al., 2012; Stefek et al., 2008).
Artichoke (Cynara cardunculus L.; Asteraceae family) is grown
mainly in Italy, Spain, and Peru, and its flesh leaves (bracts) are cooked
and eaten worldwide. The artichoke leaves were investigated for their
beneficial effects (mainly due to polyphenolic compounds) such as
inhibition of the biosynthesis of cholesterol, induction of choleresis,
mobilization of energy reserves, as well as their antibacterial and hepa-
toprotective actions (Gabbay, 2004; Zhu, Zhang, & Lo, 2004). Arti-
choke extracts are also recognized for their hypoglycemic effect and
beneficial effect on lipid metabolism of diabetic rats (Hosseini,
Mousaei, & Tavakoli, 2015; Magied, Hussien, Zaki, & Said, 2016;
Salem et al., 2017).
Potential effects of artichoke leaf extract on sorbitol content in
tissues exposed to hyperglycemia have not been thoroughly investi-
gated. The aim of this work was to elucidate the effect of artichoke
leaf extract and several compounds of its content on high‐glucose‐
induced oxidative stress and on sorbitol accumulation in tissues
exposed to high glucose, which are the potential mechanisms leading
to the development of chronic diabetic complications.
2 | MATERIALS AND METHODS
2.1 | Materials
The DMEM low glucose (5.5 mM) with sodium pyruvate (110 mg/L)
was purchased from Biosera (Nuaille, France). The penicillin–strepto-
mycin mixture was purchased from Lonza (Verviers, Belgium). Phos-
phate‐buffered saline (PBS), fetal bovine serum, dimethyl sulfoxide
(DMSO), β‐NAD+, diaphorase, resazurin, M‐199 medium (M 3769),
D‐glucose, chlorogenic acid, caffeic acid, quinic acid, luteolin, and
HClO4 were purchased from Sigma‐Aldrich (Germany). 2,7‐
dichlorodihydrofluorescein diacetate (DCFH‐DA) was purchased from
Cayman Chemical (Ann Arbor, MI, USA). The WST‐1 assay kit and the
sorbitol dehydrogenase were obtained from Roche Diagnostics
(Mannheim, Germany).
2.2 | Extract
The water infusion of artichoke leaves (C. cardunculus L., Asteraceae
family [European Pharmacopoeia 8th edition]) from material pur-
chased from Fytopharma (Malacky, Slovak Republic) was prepared
according to the Pharmacopoeia Bohemoslovaca 4. It was lyophilized
at −53 °C and 0.043 Pa in a SCANVAC CoolSafe freeze dryer
(LaboGene, Lynge, Denmark) according to the instructions.
ET AL.
2.3 | Cell culture
The mouse myoblast cell line C2C12 was obtained from the European
Collection of Cell Cultures (Salisbury, UK). Cells were maintained in
DMEM medium supplemented with 10% heat‐inactivated fetal bovine
serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in an
incubator with humidified atmosphere and flow of 5% CO2. The cells
were passaged approximately twice a week.
2.4 | Animals
The lenses removed from the experimental rats in the Department of
Pharmacology and Toxicology of the Faculty of Pharmacy of the
Comenius University in Bratislava, Slovakia, were designated for other
experiments. All experimental procedures, involving the use of exper-
imental animals, were approved by the local Ethical Committee and
the State Veterinary and Food Administration of the Slovak Republic.
The investigation conforms to the Guide for the Care and Use of Lab-
oratory Animals: Eighth Edition (2010) published by the US Committee
for the Update of the Guide for the Care and Use of Laboratory Ani-
mals, National Research Council, and to the EU adopted Directive
2010/63/EU of the European Parliament and of the Council on the
protection of animals used for experimental and other scientific
purposes.
2.5 | In vitro analysis of cytotoxicity and cell
proliferation
The effects of the compounds tested on the activity of mitochondrial
dehydrogenases of cells were assessed using the reduction of tetrazo-
lium salt WST‐1. C2C12 cells were seeded (18,000 cells/100 μl/well)
into a 96‐well plate. After 24 hr, different concentrations of the sam-
ples tested were added. The samples were dissolved in cultivation
medium or in DMSO and medium in case of luteolin. The final concen-
tration of DMSO never exceeded 0.5%. Following 24‐hr incubation, a
WST‐1 assay kit was used according to the manufacturer's instruc-
tions to determine cytotoxicity. The amount of formazan created
(which corresponds to the number of viable and metabolically active
cells) was calculated as a percentage of the control cells which were
set as 100%.
2.6 | Determination of intracellular oxidative stress
under high‐glucose conditions
The generation of reactive oxygen species (ROS) was assessed using a
DCFH‐DA probe. C2C12 cells were seeded in DMEM low glucose
medium (5.5 mM) into a black 96‐well plate (18,000 cells/100 μl/well).
After 24‐hr incubation, the test samples were added. After 1 hr, the
glucose dissolved in PBS or pure PBS was added for increasing glucose
concentration to 55 mM or retaining normal glucose conditions
(5.5 mM). After 3.5‐hr incubation, DCFH‐DA (10 μM) dissolved in
the medium was introduced. After further 30 min, the intracellular
fluorescence was measured (λ [excitation/emission] = 480/530 nm)
in Fluostar Omega Microplate Reader (BMG Labtech).
ŠUŠANÍKOVÁ ET AL.
2.7 | Eye lenses sorbitol assay
The animals in light ether anesthesia were sacrificed by bleeding the
cervical artery, and the eye globes were excised. The lenses were
quickly dissected, rinsed with PBS, and weighed. The lenses were
incubated at 37 °C in tubes (one lens per tube) in M‐199 medium at
pH 7.4, bubbled with pneumoxide (5% CO2 and 95% O2), and with
the compounds tested, they were soluted in the medium. After
30 min, glucose was added to the tubes to the final concentration of
50 mM, except the negative control. Then the incubation was contin-
ued following 3 hr at 37 °C with occasional (in about 30‐min intervals)
bubbling of the mixture for approximately 30 s periods with
pneumoxide. The incubation was terminated by cooling the mixtures
in an ice bath, followed by washing the lenses three times with
ice‐cold PBS (1 ml). Short‐term cultivations were preferred to avoid
substantial permeability changes of the eye lenses. The washed lenses
were kept deep‐frozen for sorbitol determination, which was per-
formed as described before (Juskova et al., 2009). In brief, the frozen
lenses were let to melt at ambient temperature. Then distilled water
(0.2 ml/one lens) was added. The lenses were disrupted by a glass
rod. The tissue material stuck to the rod was washed out twice with
distilled water (0.1 ml) back into the tube, and the suspension was
ultrasounded for 5 min. Then ice cold HClO4 (9%, 0.4 ml) was added
and mixed thoroughly. The mixture was ultrasounded for further
5 min and then kept on ice for 30 min to let proteins precipitate.
The precipitated protein was spun off (15 min at 3,000 rpm) at 4 °C.
The supernatant was neutralized with 60 μl of concentrated K2CO3
(4 M). The neutralized supernatant was used for determination of
the concentration of sorbitol by modified enzymatic analysis (Mylari
et al., 2003). The amount of sorbitol in nanomole per gram of lens
wet weight in each sample was determined by comparison with a
linear regression of sorbitol standards.
2.8 | Statistical analysis
The data were graphed as means ± standard error of mean. Compari-
sons among groups were made using the Student t test (calculated by
website in‐silico.net/tools/statistics/t‐test) with equal variance.
3 | RESULTS AND DISCUSSION
The artichoke leaves are well known for their beneficial therapeutic
effects in enhancing lipid metabolism, choleresis, and hepatic func-
tions. In several studies, the extracts from artichoke leaves showed
also decreasing activities of glucose level and beneficial effect on lipid
metabolism of diabetic rats (Hosseini et al., 2015; Magied et al., 2016;
Salem et al., 2017) as well as effective AKR1B1 and RL‐AKR inhibi-
tions (Miláčková et al., 2017). Human AKR1B1 (and rat equivalent
RL‐AKR) is the first enzyme of the polyol pathway that reduces
glucose to sorbitol more intensively under diabetic conditions.
Increased flow of the polyol pathway could cause osmotic stress due
to the created sorbitol and oxidative stress as result of consumed
cofactor NADPH, important for glutathione. For a long time, many
in vitro and in vivo studies have presented inhibitors of AKR1B1 and
antioxidants like potential therapeutic agents against development of
3
chronic diabetic complications such as cataract, diabetic nephropathy,
neuropathy, and others (Coudert et al., 1994; Juranek et al., 2009;
Karasu et al., 2012; Stefek et al., 2008).
The aim of this experimental work was to expand the findings
about potential therapeutic effects of artichoke leaf extract on chronic
diabetic complications. The effects of artichoke extract on models of
cell culture and isolated organ under high‐glucose conditions were
evaluated. Phenolic compounds present in the extract were investi-
gated to better understand which particular constituent is responsible
for the effect. However, the concentrations of the phenolic com-
pounds used are greater than their concentrations in raw extract.
Moreover, many derivatives of caffeic acid and quinic acid occur in
the extract. These derivatives could possess similar effects as their
parental molecules, or they could be metabolized into parental
molecules.
The cell line C2C12, mouse myoblasts, was chosen for these
experiments. Muscle cells are very active in glucose transport and
metabolism. The cytotoxicity of the substances tested and their ability
to inhibit proliferation were investigated by WST‐1 assay (Figure 1).
The amount of formazan formed, by reduction of the tetrazolium salt
WST‐1, corresponds directly to the number of viable cells with active
mitochondrial reductases. The concentrations of artichoke extract up
to 50 μg/ml were significantly cytotoxic. None of the participating
compounds tested did show cytotoxic effects at 50‐μM concentra-
tion. The concentrations of the substances tested, which did not
decrease the cell count (2 and 6 μg/ml for artichoke extract and 2
and 6 μM for pure compounds), were chosen for subsequent studies
on cell culture.
The muscle cells C2C12 were incubated with the test samples in
two concentrations (Figure 2) in the models of normoglycemia
(5.5 mM; Figure 2a) and hyperglycemia (55 mM glucose; Figure 2b)
for 4 hr. Oxidative stress was determined by DCFH‐DA fluorescence
probe. After 4 hr, the intracellular content of ROS in the cells incu-
bated in high‐glucose conditions was significantly (p < 0.01) increased
to 129.81% ± 14.13% (positive control) contrary to normal glucose
conditions (negative control; 100%). In a similar study in C2C12 cells,
ROS level increased to about 180% in high‐glucose conditions
(100 mM glucose) compared with normal glucose conditions after
2 hr of incubation (Kang et al., 2015).
In normal glucose conditions, incubation with the samples tested
significantly decreased basal ROS concentration by 15%–30%, except
for 6 μM quinic acid (10%) and 6 μM luteolin (0%). Of phenolic com-
pounds, luteolin showed weaker antioxidant capacity confirmed in
in vitro study (Rice‐Evans, Miller, & Paganga, 1996). In high‐glucose
conditions, incubation with all samples tested protected cells against
oxidative stress, along with significant decrease of ROS concentration
in the range of 20%–40%.
There are several studies that determined intracellular level of
ROS after short‐term or long‐term incubations with high glucose
(Busik, Mohr, & Grant, 2008; Mousavi, Tayarani, & Parsaee, 2010;
Wu et al., 2004). The studies show differences among cell types in
the sensitivity to glucose toxicity. A number of parameters could be
affected by glucose toxicity, like expression and regulation of different
types of glucose transporters and hexokinase isoforms, the rate of
glycolysis versus oxidative phosphorylation, antioxidant protection
4 ŠUŠANÍKOVÁ ET AL.

FIGURE 1 Comparison of the viabilities (assessed by mitochondrial reduction of WST‐1) of C2C12 cells after 24‐hr treatment with (a) artichoke
leaf extract, (b) caffeic acid, (c) chlorogenic acid, (d) quinic acid, and luteolin. The values are expressed as percentage of control (100%) and are the
means ± standard error of three independent experiments. * indicates p < 0.05, ** p < 0.01, and *** p < 0.001 (vs. untreated control) [Colour figure
can be viewed at wileyonlinelibrary.com]

level, and others. In any case, the test samples showed significant
antioxidative effects under normal and high‐glucose conditions and
could be used for prevention of oxidative damage caused by glucose
toxicity. Artichoke leaf extracts exhibited antioxidative properties in
a number of studies (Brown & Rice‐Evans, 1998; Miccadei et al.,
2008) due to their phenol content. The well‐known antioxidants
caffeic acid, chlorogenic acid, quinic acid, and luteolin showed signifi-
cant antioxidative activities also in this study. The effect of ROS on
muscle cells could have hormetic nature. In muscles, ROS may trigger
different signaling pathways leading to diverging responses, from
adaptation to cell death. Whether a “positive” or “negative” response
will prevail, depends on some variables such as the persistence of
ROS flow, the antioxidant capacity and the energy status of muscle
cells and others (Barbieri & Sestili, 2012).
The next experiment was performed at the level of isolated organ
using rat eye lens, the organ containing high concentration of aldose
reductase protein (Lee, Chung, & Chung, 1995), in hyperglycemic
surroundings. It is a model of development of chronic diabetic compli-
cations in vitro and has been conventionally used (Chylack, Henriques,
& Tung, 1979; Moghaddam, Kumar, Reddy, & Ghole, 2005). Aldose
reductase is the first enzyme of the polyol pathway which is believed
to have a key role in the development of diabetic cataract and other
chronic diabetic complications. As shown in Figure 3, significantly
increased sorbitol levels were recorded in the isolated rat lenses
incubated with high‐glucose concentration (50 mM; positive control)
in comparison with controls incubated without glucose (negative
control). This reflects an increased flux of glucose through the polyol
pathway. Similarly, threefold increase of sorbitol level in the eye lenses
incubated with high glucose for 4 hr was observed in a previous study
(Milackova et al., 2015). Sorbitol production (determined by fluores-
cent method) was significantly inhibited by all test samples (Art, ChA,
and CA in two concentrations). The findings indicate that all three
samples are readily taken up by eye lens cells where they interfere
with cytosolic aldose reductase. The content compounds of artichoke
leaf extract, caffeic acid, and chlorogenic acid as well as several other
caffeoyl derivatives of quinic acid have been recognized as effective
inhibitors of the first enzyme of the polyol pathway (Jang et al.,
2010; Kim et al., 2011; Yoon, Lee, Kim, Suh, & Lim, 2013), and thus,
they may contribute to the sorbitol decreasing effect of the extract.
To our knowledge, artichoke extracts have never been studied in the
ŠUŠANÍKOVÁ ET AL. 5

FIGURE 2 Effect of test samples on the

generation of ROS in C2C12 cells in the model
of (a) normoglycemia (5.5 mM of glucose) and
(b) hyperglycemia (55 mM of glucose). The
cells were pretreated with the test samples or
with vehicle only (controls) for 1 hr. Then
glucose or PBS only was added to reach the
demanded glucose conditions. After 3.5‐hr
incubation, DCFH‐DA was introduced. After
further 30 min, the intracellular fluorescence
of DCF was measured, which determined the
intracellular ROS level. The values are
expressed as percentage of negative control
(Neg Ctrl; 100%) in the case of (a) or positive
control (Pos Ctrl; 100%) in the case of (b) and
are means ± SE of three independent
experiments. * indicates p < 0.05, ** p < 0.01,
and *** p < 0.001 (vs. untreated controls). Art:
artichoke leaf extract; CA: caffeic acid; ChA:
chlorogenic acid; QA: quinic acid; Lut: luteolin;
PBS: phosphate buffered saline; DCF: 2′‐7′‐
dichlorofluorescein; DCFH‐DA, 2,7‐
dichlorodihydrofluorescein diacetate; ROS:
reactive oxygen species; SE: standard error

FIGURE 3 Effects of Art and two phenolic

compounds on sorbitol accumulation in
isolated rat lenses cultivated with high
glucose. The lenses were incubated in medium
with the samples tested for 30 min at 37 °C.
Then glucose was added in the final
concentration of 50 mM, except for negative
control (Neg Ctrl), and incubation continued
for the following 3 hr. The sorbitol level in the
lenses was determined by enzymatic analysis.
The values are expressed as nmol/g of lens
and are the means ± SE of three independent
experiments. * indicates p < 0.05, ** p < 0.01,
and *** p < 0.001 (vs. positive control [Pos
Ctrl]). Art: artichoke leaf extract; CA: caffeic
acid; ChA: chlorogenic acid; SE: standard
deviation

relation to chronic diabetic complications and our results provide new
information to this field. A few in vivo studies of cataract were done
with phenolic compounds. Chlorogenic acid was reported to act pre-
ventively against development of sugar cataract on the galactose‐fed
rat model (Kim et al., 2011). Caffeic acid phenethyl ester effectively
suppressed sodium‐selenite‐induced cataract formation in rats
(Doganay et al., 2002).
In conclusion, the present study extends our previous work on bio-
logical activities of artichoke leaf extract (Miláčková et al., 2017) reveal-
ing antioxidant activity in cultured C2C12 cells exposed to high glucose.
In addition, the extract effectively inhibited accumulation of sorbitol in
the rat eye lenses exposed to high glucose ex vivo. On balance, the
artichoke leaf extract represents a prospective preventive agent of
chronic diabetic complications in view of preclinical and clinical studies.
6 ŠUŠANÍKOVÁ
ACKNOWLEDGMENTS
This work was supported by grants VEGA 1/0359/18, VEGA 1/0561/
18, VEGA 1/0290/16, VEGA 2/0041/15 (Agentúra Ministerstva
Školstva, Vedy, Výskumu a Športu SR), FaF UK/6/2016 (Faculty of
Pharmacy, Comenius University in Bratislava), APVV‐15‐0308,
APVV‐16‐0207 (Ministerstvo školstva, vedy. výskumu a športu
Slovenskej republiky), and IGA VFU 313/2018/FaF (J.H., Veterinární
a Farmaceutická univerzita Brno). Dr. Zuzana Kmecová, Dr. Eszter
Bögy, and Assoc. Prof. Dr. Karel Šmejkal are acknowledged for their
contributions and additional material.
CONF LICT OF INT E RE ST S
The authors declare that they have no conflict of interest.
ETHI CAL APPROVAL
All applicable international, national, and/or institutional guidelines for
the care and use of animals were followed.
ORCID
Ivana Šušaníková http://orcid.org/0000-0003-2365-8852
Jan Hošek http://orcid.org/0000-0003-0975-1671
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How to cite this article: Šušaníková I, Balleková J, Štefek M,
Hošek J, Mučaji P. Artichoke leaf extract, as AKR1B1 inhibitor,
decreases sorbitol level in the rat eye lenses under high glu-
cose conditions ex vivo. Phytotherapy Research. 2018;1–7.
https://doi.org/10.1002/ptr.6174