ANTIOXIDANT PROPERTIES OF METHANOLIC EXTRACTS FROM

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 2 pp. 433ñ438, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
ANTIOXIDANT PROPERTIES OF METHANOLIC EXTRACTS FROM
THE SHOOTS AND ROOTS OF pRi-TRANSFORMED PLANTS OF
REHMANNIA GLUTINOSA LIBOSCH
EWELINA PI•TCZAK1*, MARTA D BSKA2, BOGDAN KONTEK2, BEATA OLAS2
and HALINA WYSOKI—SKA1
Department of Biology and Pharmaceutical Botany, Medical University of Lodz,
MuszyÒskiego 1, 90-151 £Ûdü, Poland
2
Department of General Biochemistry, Faculty of Biology and Environmental Protection,
University of Lodz, Pomorska 141/3, 90-236 £Ûdü, Poland
1
Abstract: The antioxidant activity of methanolic extracts derived from shoots (HR-shoots) and roots (HRroots) of pRi-transformed Rehmannia glutinosa plants were determined. The activity was indicated by the ability of the plant extracts to inhibit superoxide anion (O2-∑) generation and thiobarbituric acid reactive substances
(TBARS) production in resting blood platelets and platelets activated by thrombin. The strongest activity was
exhibited by the HR-shoot extract (50 µg/mL). The present study also examines the antioxidant properties of
the plant extracts against human plasma lipid peroxidation induced by strong biological oxidants: hydrogen peroxide (H2O2) and H2O2/Fe. The study shows that extracts from transformed R. glutinosa plants may be a promising source of natural antioxidants, which would be valuable in various cardiovascular diseases. The extracts
may also protect lipids against oxidative modifications.
Keywords: antioxidant property, Rehmannia glutinosa, pRi-transformed plants
cells and by the release of inflammatory mediators
upon activation. Shoot and root extracts from seedderived R. glutinosa plants have been found to have
in vitro antiplatelet activity by Piπtczak et al. (6),
who report that only root extracts exhibit weak
activity at the highest concentration used (50
µg/mL).
The aim of the present study was to evaluate
the direct effects of methanolic extracts from shoots
and roots of pRi-transformed R. glutinosa plants on
superoxide anion (O2-∑) generation and on non-enzymatic lipid peroxidation and arachidonic acid
metabolism by blood platelets in vitro. In addition, it
also aims to determine the antioxidant activity of the
plant extracts against the effect of a strong biological oxidant, i.e., hydrogen peroxide (H2O2) or
H2O2/Fe (a hydroxyl radical donor), on human plasma lipid peroxidation. The experimental models
used in the study were similar to the reactions which
take place in human plasma under oxidative stress
conditions.
Rehmannia glutinosa Libosch ñ a member of
the Orobanchaceae family (1) is naturally distributed in China, Japan and Korea. It has been one of
the most frequently used medicinal plants in
Traditional Chinese Medicine for thousands of
years. The underground parts (Rehmanniae Rhizoma) are recognized as pharmaceutically useful,
being described in the Chinese Pharmacopoeia (2)
as possessing antianemic, antiinflammatory, antisenescence, antitumor and hypoglycemic properties
(3, 4). R. glutinosa plants have previously been
regenerated from hairy roots transformed by
Agrobacterium rhizogenes (pRi-transformed
plants), and these plants demonstrated increased
production of iridoid (catalpol, aucubin, catalposide, loganin) and phenylethanoid (verbascoside)
glycosides than untransformed plants derived from
seeds (5).
Blood platelets play an important role, not only
during hemostasis, but also in modulating immune
responses through their interaction with immune
* Corresponding author: e-mail: [email protected]
433
434
EWELINA PI•TCZAK et al.
Preparation of extracts
The shoots and roots (500 mg) were
lyophilized, powdered and extracted with methanol
(40 mL). Methanolic extracts were prepared as
described earlier (7). Extraction yields (% w/w)
were calculated by the following formula: weight of
the dried extract (g)/weight of the original sample
(g) ◊ 100%. The extract yields were 40.2% for the
shoot extracts and 61.6% for the root extracts.
Blood platelet isolation
Peripheral blood was collected from nonsmoking men and women into ACD solution (citric
acid/citrate/dextrose; 5 : 1; v/v; blood/ACD). The
protocol was approved by the Committee for
Research on Human Subjects, University of Lodz
(reference: 2/KBBN-U£/III/2014). Platelet-rich
plasma (PRP) was prepared by centrifugation of
fresh human blood at 250 ◊ g for 10 min at room
temperature. Platelets were then sedimented by centrifugation at 500 ◊ g for 10 min at room temperature. The platelet pellet was washed twice with
Tyrodeís buffer containing 10 mM HEPES, 140 mM
NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM NaHCO3
and 10 mM glucose (pH 7.4), and the platelets were
suspended in Tyrodeís buffer. The concentration of
platelets in platelet suspensions estimated spectrophotometrically (8) was about 5 ◊ 108/mL.
Suspensions of blood platelets were incubated
with plant extracts at final concentrations of 0.5-50
µg/mL (15 min, at 37OC) with or without the addition of thrombin (5 U/mL, 5 min 37OC).
Chemicals
Cytochrome c, dimethyl sulfoxide (DMSO)
5,5í-dithio-bis-(2-nitrobenzoic acid) (DTNB), thiobarbituric acid (TBA), H2O2 were purchased from
Sigma (St. Louis, MO., USA). All other reagents
were of analytical grade and were provided by commercial suppliers. The stock solution of plant
extracts used for testing was made in 50% DMSO.
The final concentration of DMSO in samples was
lower than 0.05% and its effects were determined in
all experiments.
Plasma isolation
Fresh human plasma was obtained from medication free, regular donors at the blood bank (£Ûdü,
Poland); the protocol was passed by the Committee
for Research on Human Subjects of the University
of Lodz, (reference: 2/KBBN-U£/III/2014).
Plasma was incubated with:
● plant extract at final concentrations of 0.5-50
µg/mL (15 min, at 37OC);
● plant extract at final concentrations of 0.5-50
µµg/mL plus 2 mM H2O2 (15 min, at 37OC);
MATERIALS AND METHODS
Plant material
Shoots (HR-shoots) and roots (HR-roots) originating from 12-month-old in vitro regenerated
Rehmannia glutinosa Libosch transformed plants
growing in pots in a greenhouse were used for the
experiments. The plants were derived from adventitious shoots which spontaneously appeared on the
surface of hairy roots (line RS-2) infected by
Agrobacterium rhizogenes strain A4 as described
earlier (5).
Table 1. Inhibitory effects of extracts from shoots and roots of transformed R. glutinosa plants (0.5-50 µg/mL,
15 min, 37OC) on superoxide anion generation in resting blood platelets and platelets activated by thrombin (5
U/mL, 5 min, 37OC).
Inhibition of O2-∑
generation in resting
blood platelets (%)
Inhibition of O2-∑
generation in blood platelets
activated by thrombin (%)
0.5
9.1a ± 3.6
23.0bd ± 2.6
5.0
18.7 ± 6.2
32.1c ± 5.1
50
37.8b ± 6.8
42.5e ± 5.5
0.5
10.6a ± 4.2
19.6b ± 2.8
5.0
16.2 ± 6.9
26.6cd ± 4.6
50
2.5ab ± 10.2
1.2cd ± 7.2
Extract
concentration
(µg/mL)
Shoot extract
a
Root extract
a
Data represent the means of 5 - 7 donors ± standard error (SE). Values with the same letter within a column are
not significantly different according to the Kruskal-Wallis test (p ≤ 0.05).
Antioxidant properties of methanolic extracts from the shoots and roots of...
435
Table 2. Inhibitory effects of extracts from shoots and roots of transformed R. glutinosa plants (0.5-50 µg/mL,
15 min, 37OC) on the level of TBARS in resting blood platelets and platelets activated by thrombin (5 U/mL, 5
min, 37OC).
Extract
concentration
(µg/mL)
Inhibition of TBARS
production in resting blood
platelets (%)
Inhibition of TBARS
production in blood platelets
activated by thrombin (%)
0.5
4.9a ± 2.6
21.4b ± 5.5
5.0
7.5 ± 2.6
27.7c ± 2.9
50
15.0b ± 5.3
43.9de ± 11.3
0.5
4.4a ± 2.7
21.2b ± 7.9
5.0
9.1 ± 4.5
38.3d ± 4.9
50
20.9b ± 10.4
54.5e ± 4.2
Shoot extract
a
Root extract
a
Data represent the means of 3 - 4 donors ± standard error (SE). Values with the same letter within a column are
not significantly different according to the Kruskal-Wallis test (p ≤ 0.05).
●
plant extract at final concentrations of 0.5-50
µg/mL plus 4.7 mM H2O2/3.8 mM Fe2SO4/2.5
mM EDTA (15 min, at 37OC).
Superoxide anion measurement
The cytochrome c reduction method was used
as described earlier (9) to test O2-∑ generation in controls and in blood platelets incubated with tested
compounds. Briefly, an equal volume of modified
Tyrodeís buffer containing cytochrome c (160 µM)
was added to a platelet suspension. After incubation,
the platelets were sedimented by centrifugation at
2000 × g for 5 min and the supernatants were transferred to cuvettes. Any reduction in cytochrome c
was measured spectrophotometrically at 550 nm. To
calculate the molar concentration of O2-∑, the molar
extinction coefficient for cytochrome c was taken as
18,700 M-1 cm-1.
Lipid peroxidation measurement
Lipid peroxidation was quantified by measuring the concentration of thiobarbituric acid reactive
substances (TBARS). Incubation of plasma (control,
plant extract and H2O2 ñ or H2O2/Fe -treated plasma)
was stopped by cooling the samples in an ice bath.
Samples of plasma were transferred to an equal volume of 20% (v/v) cold trichloroacetic acid in 0.6 M
HCl and centrifuged at 1200 ◊ g for 15 min. Clear
supernatant was mixed with 0.12 M thiobarbituric
acid in 0.26 M Tris at pH 7.0 in a ratio of 5 : 1 by
volume and immersed in a boiling water bath for 15
min. Absorbance was measured at 532 nm
(Spectrophotometer UV/Vis Helios alpha Unicam)
(10). The TBARS concentration was calculated
using the molar extinction coefficient (ε = 156,000
M-1 cm-1).
Data analysis
In order to eliminate uncertain data, the QDixon test was performed. A total of 5-7 independent experiments were performed and the results
were expressed as the mean ± standard error (SE).
Statistically significant differences between results
were also identified by applying the Kruskal-Wallis
test at a significance level of p ≤ 0.05 using STATISTICA 10 (StatSoft) software.
RESULTS
Extracts from shoots and roots of pRi-transformed R. glutinosa plants at tested concentrations
(0.5 ñ 50 µg/mL) reduced the generation of O2-∑ in
resting blood platelets and platelets activated by
thrombin in vitro (Table 1). The presented results
indicate that the shoot extracts were more effective
than the root extracts (at p ≤ 0.05). The inhibition of
O2-∑ generation was found to be significantly higher
in activated (thrombin-treated) blood platelets than
the untreated resting platelets (p ≤ 0.05) (Table 1).
The greatest inhibition (42.5 %) was observed in the
HR-shoot extract at a concentration of 50 µg/mL, for
blood platelets activated by thrombin (Table 1).
In addition, the TBARS level was measured as
non-enzymatic lipid peroxidation in resting blood
platelets and as enzymatic lipid peroxidation of arachidonic acid in blood platelets stimulated by thrombin.
After a 15 min pre-incubation of platelets with shoot
and root extracts of pRi-transformed R. glutinosa
436
EWELINA PI•TCZAK et al.
plants at tested concentrations (0.5ñ50 µg/mL), the
amount of TBARS in resting platelets and thrombinactivated blood platelets was seen to diminish. The
action of two tested extracts was concentration
dependent. In the presence of the highest extract concentrations (50 µg/mL), TBARS production in activated platelets was reduced by about 44% in the shoot
extract and 55% in the root extract (Table 2).
As shown in Table 3, the extracts from the
shoots and roots of R. glutinosa transformed plants
reduced also plasma lipid peroxidation induced by
H2O2 or H2O2/Fe. The reduction in TBARS level in
plasma treated with the 50 µg/mL extracts from pRitransformed plants in the presence of H2O2 reached
about 50% for the shoot extract and about 30% for
the root extract (Table 3). No tested extracts had any
effect on plasma lipid autoperoxidation in vitro (data
not presented).
In control experiments, DMSO (the solvent)
added to platelet suspensions at a final concentration
below 0.05% did not influence platelet activation in
the studied assays (data not shown).
DISCUSSION
In this study, we report for the first time the
antioxidant activity of methanolic extracts from
shoots and roots derived from pRi-transformed R.
glutinosa plants. The results indicate that the activity of the R. glutinosa extracts were 33% higher
than those observed earlier for extracts from the
untransformed organs (shoots and roots) originated
from seed-derived plants (6). Piπtczak et al. (6)
reported that 50 µg/mL of root extract was sufficient to inhibit the generation of O2-∑ in blood
platelets. Shoot extracts were inactive in tested concentrations (6). It was also earlier reported that
seed-derived shoot extract was also able to protect
plasma lipid against peroxidation caused by hydrogen peroxide. Root extracts of the plants were inactive in the assays (6). The results of the present
study indicate that shoot and root extracts from R.
glutinosa transformed plants exhibit stronger activity against plasma lipid peroxidation than those of
untransformed plants. These superior biological
activities of the extracts could be caused by higher
levels of some bioactive iridoid (catalpol, aucubin,
loganin) and phenylethanoid (verbascoside) glycosides in the transformed organs than in the shoots
and roots of seed-derived plants. Piπtczak et al. (5)
reported that transformed roots produce almost
twice the amount of catalpol and loganin compared
to the roots of untransformed plants. Another iridoid, aucubin, was not detected in the untransformed plants, although low amounts of the compound (0.05 mg/g of dry weight - d.w.) were found
in roots of transformed plants (5). Also, the contents of verbascoside in transformed shoots (21.7
mg/g d.w.) and roots (4.6 mg/g d.w.) were found to
be higher than in the shoots and roots of untransformed R. glutinosa (18.1 mg/g d.w. and 2.8 mg/g
d.w., respectively) (5).
The experimental models used in the present
study are similar to the reactions taking place in
human blood platelets and human plasma. It has
been proposed that reactive oxygen species (ROS)
production is a critical signal regulating blood
platelet activity. Some natural substances with different biological activities, including antioxidant
and antiplatelet activities, may modify this
Table 3. Inhibitory effects of extracts from leaves and roots of transformed R. glutinosa plants (0.5-50 µg/mL,
15 min, 37OC) on plasma lipid peroxidation induced by H2O2 and plasma lipid peroxidation induced by
H2O2/Fe.
Extract
concentration
(µg/mL)
Inhibition of TBARS
production induced by H2O2
(%)
Inhibition of TBARS
production induced by
H2O2/Fe (%)
0.5
44.7b ± 11.8
13.1b ± 4.6
5.0
53.4b ± 10.0
19.9c ± 4.5
50
47.3 ± 11.5
20.9c ± 5.5
0.5
34.9b ± 8.6
11.3b ± 5.5
5.0
40.5b ± 7.6
11.9b ± 6.5
50
32.5 ± 6.8
15.9c ± 6.3
Shoot extract
b
Root extract
b
Data represent the means of 5 donors ± standard error (SE). Values with the same letter within a column are
not significantly different according to the Kruskal-Wallis test (p ≤ 0.05).
Antioxidant properties of methanolic extracts from the shoots and roots of...
process. Hence, the inhibitory effect of the tested
extracts on ROS generation in platelets activated
by thrombin may be involved in inhibition of various steps of platelet activation in blood platelets.
Thrombin, as serine protease, evokes biological
responses from a variety of cells such as blood
platelets, megakaryoblasts, endothelial cells,
monocytes or smooth muscle cells. All of the
effects of thrombin are mediated by receptors on
the cell surface (11). The biological effects of
thrombin on blood platelets include platelet aggregation, secretion of various compounds from
dense and α granules, C2+ mobilization, arachidonate and phosphoinositide metabolism (11). Some
reports indicate that low concentrations of thrombin have antithrombotic and inflammatory properties (12, 13).
Moreover, during platelet activation, the generation of reactive oxygen species (ROS), mainly
superoxide anions, also occurs. ROS may potentiate the response of platelets and may also act as
signaling radicals and cause blood platelet activation. The main source of ROS is the arachidonate
pathway, which is dependent on cyclooxygenase
or 12-lipooxygenase, the glutathione cycle and
metabolism of phosphoinositides. ROS, including
superoxide anions, may regulate blood platelet
function by decreasing the bioavailability of nitric
oxide due to peroxynitrite formation (14). Our
results indicate that in the presence of the highest
concentration of the extract from pRi-transformed
R. glutinosa (50 µg/mL), TBARS generation in
stimulated platelets was inhibited by about 44% in
the shoot extract and 55% in the root extract
(Table 2).
Our findings are in accordance with those
reported earlier by Gao et al. (15) and Liu et al.
(16) who showed that R. glutinosa can inhibit lipid
peroxidation, and therefore, possesses an antisenescence activity. Additionally, Chang et al. (17)
and Chae et al. (18) reported the favorable influence of R. glutinosa extract on the cardiovascular
system (blood pressure and cardiac muscle cells).
It seems possible that the extracts tested in our
study may play a role in modulating platelet adhesion by interfering with the metabolism of arachidonic acid, in which cyclooxygenase or lipoxygenase play a role. After 15-min preincubation of
platelets with the extracts from R. glutinosa the
amount of TBARS (representing the non-enzymatical peroxidation of arachidonic acid) in thrombinstimulated platelets was reduced (Table 2), and it is
possible that the extracts from R. glutinosa might
also inhibit cyclooxygenase or lipoxygenase activ-
437
ity in these cells. As reported by Park et al. (19)
and Anauate et al. (20), some iridoids, including
those present in the tested extracts of R. glutinosa,
showed inhibition of cyclooxygenase-1/2 activity
in human erythroleukemia cells (aucubin) a murine
macrophage cell line (loganin) and a whole-blood
assay (harpagoside). Of the phenylethanoids, verbascoside, also detected in the extracts of R. glutinosa (5), demonstrates anti-inflammatory activity
in several models, including inhibition of
cyclooxygenase-2 activity (20-23). Based on these
observations, the effect of the plant extracts on
cyclooxygenase activity will be the subject of
future studies.
CONCLUSIONS
The greater inhibition of O2-∑ generation in
blood platelets described in the present study, as
well as the higher degree of antioxidant activity
observed in transformed organ extracts compared to
the organs of untransformed plants, indicates that
the Agrobacterium rhizogenes-mediated transformation had a positive influence, not only manifested by
higher biomass accumulation and iridoid and
phenylethanoid production, as reported by Piπtczak
et al. (5), but also by the increased antioxidant activity of transformed R. glutinosa plants. The study
shows that extracts from transformed R. glutinosa
plants may not only be a promising source of natural antioxidants, but also of compounds demonstrating antiplatelet activity, which would be valuable in
various diseases associated with oxidative stress or
those demonstrating blood platelet hyperactivity in
cardiovascular diseases. It may also be an important
cofactor of anti-inflammatory reactions. On the
basis of the present experiments, we indicate that
extracts from R. glutinosa may inhibit free radical
production and protect lipids against oxidative modifications. The concentrations of the R. glutinosa
extracts used in our experiments (5-50 µg/mL) correspond to the range of physiological compounds
after supplementation or dietary intake, and so may
be potentially therapeutically useful in the prevention of several diseases.
Acknowledgment
This work was supported by the University of
Lodz under grant no. 506/1136.
Declaration of interest
The authors declare no conflicts of interest.
438
EWELINA PI•TCZAK et al.
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Received: 4. 11. 20115