Posters P5.1 P5.2 Poster Session - Acta Biochimica Polonica

P5.25
Saponinis from the leaves
of Knautia arvensis
Jarosław Mołdoch 1 , Barbara Szajwaj 1 , Dariusz
Jędrejek 1 , Mariusz Kowalczyk 1 , Milena Masullo 2 , Sonia
Piacente 2 , Wiesław Oleszek 1 , Anna Stochmal 1
1 Institute of Soil Science and Plant Cultivation State Research Institute,
Department of Biochemistry, Pulawy, Poland; 2 Università degli Studi
di Salerno, Dipartimento di Scienze Farmaceutiche e Biomediche, Via
Ponte Don Melillo, Fisciano, Italy
e-mail: Jaroslaw Moldoch
The 6FP European Union programme (Rumen-up) was
aimed at the development of a practical, natural alternatives
to antibiotic growth promoters used in animal feed.
In this study Knautia arvensis extract showed high anti-proteolytic
activity in ruminants. Till recently, no comprehensive
reserach on secondary metabolites of K. arvensis has
been performed and its anti-proteolitic effect could not be
correlated to any group of compounds. Methanol extract
from K. arvensis contained a wide spectrum of secondary
metabolites, especially phenolic acids, flavonoids and saponin
glycosides. This study was performed to characterize
the profile of saponins in the aerial parts of K. arvensis.
Plant material was collected in the fields near Janów Lubelski
(Poland). Crude extract of Knautia arvensis was obtained
through the extraction of dry plant material (41 g) with
70% methanol. Saponin fraction was separated from the
crude extract by Solid Phase Extraction (SPE) on C18
short column. To elute saponins, the column was washed
with 80% methanol. Crude saponins were separated on
the Gilson’s Analytical to Semi-preparative HPLC system.
Separations were performed on Atlantis Prep T3 column
(5 µm, 250 mm × 10 mm) using a isocratic elution (mobile
phase consisting of 28% acetonitrile in 0.5% acetic acid).
Column was maintained at room temperature. The separation
was completed in 40 min. at flow rate of 6 ml/min.
Structures of the isolated saponins were elucidated by
spectroscopic techniques. Mass information and fragmentations
were gathered using a Thermo LCQ Advantage
Max ion-trap mass spectrometer coupled with a Surveyor
HPLC system. Separation was performed on a 150 mm x
2.1 mm i.d., 5 µm Symmetry C18 column (Waters) using a
linear gradient from 5 to 65% of solvent B (acetonitrile)
in solvent A (water containing 0.025% formic acid), with a
flow rate of 0.4 µl/min. The mass spectrometer operated
in the negative electrospray mode with the following ion
source parameters: spray voltage 3.9 kV, capillary voltage
–47 V, tube lens offset –60 V, capillary temperature 240°C.
Nitrogen sheath and auxiliary gas flows were of 65 and 10
arbitrary units, respectively. In each scan, the peak with the
highest intensity was fragmented using collision-induced
dissociation (CID, 35% normalized collision energy).
The NMR technique was used for detailed elucidation of
structures. Analyses were done on Bruker DRX-600 spectrometer
(Bruker BioSpinGmBH, Rheinstetten, Germany)
equipped with a Bruker 5 mm TCI CryoProbeat 300 K.
All 2D NMR spectra were acquired in CD 3 OD (99.95%,
Sigma–Aldrich) and standard pulse sequences and phase
cycling were used for DQF-COSY, HSQC and HMBC
spectra. The NMR data were processed using UXNMR
software. Chemical shifts were recorded in ppm as δ relative
to tetramethylsilane (TMS) as internal standard.
Two saponins were isolated and their structures confirmed
as mazusaponin I and mazusaponin II. These compounds
were not reported in K. arvensis so far.
Poster Session 49
P5.26
Pre-exposure of Phaseolus coccineus
to methyl jasmonate as a factor
modifying copper toxicity
Agnieszka Hanaka, Małgorzata Wójcik, Sławomir
Dresler, Ewelina Nowak, Waldemar Maksymiec
Maria Curie-Skłodowska University, Institute of Biology, Department of
Plant Physiology, Lublin, Poland
e-mail: Agniwszka Hanaka
II Conference of Bioactive Plant Compounds, Puławy, Poland, 2011
Copper is an essential metal for normal plant growth and
development, although its excess is toxic. Excess copper
inhibits plant growth and impairs cellular processes such as
photosynthetic electron transport. Methyl jasmonate (MJ)
is a signaling molecule that plays a key role in the response
to external stress and in the regulation of metabolic processes.
The aim of the study was to determine the toxic
effect of copper on runner bean (Phaseolus coccineus L. cv.
Piękny Jaś) leaves after MJ pre-exposure.
Plants were cultivated hydroponically in the controlled conditions
in the growth chamber. The following 9 treatments
were used: control, 50 and 100 µM Cu, 10 µM MJ alone
for 1 and 24 h and pre-exposure to 10 µM MJ for 1 and
24 h followed by 50 and 100 µM Cu. Metal treatment was
5 days long and after this period of time the 10-day-old
plants condition was measured and presented in such parameters
as followes: the shoots mass, leaves area, electrolyte
leakage, photosynthetic pigments and photosynthetic
apparatus efficiency parameters such as: the potential photochemical
efficiency of photosystem II (Fv/Fm), vitality
index (Rfd), photochemical quenching coefficient (qP),
non-photochemical quenching (NPQ), light energy not
used for chemistry (LNU), additional energy emitted per
energy absorbed in the antenna PSII due to the closure of
a fraction of reaction centers (%X).
All Cu-treated plants showed significantly reduced biomass
and smaller leaves area. The highest leaves area occurred
in plants treated MJ alone for 24 h. The electrolyte leakage
was significantly lower in all plants 50 µM Cu-treated
and in all plants MJ-treated with exception of 100 µM Cu
with 1 h MJ exposure. It seems that MJ played a protective
role in reduction of membrane permeability and declining
integrity. Chlorophyll a+b level and chlorophyll a+b/carotenoids
ratio were the lowest in plants exposed to 100 µM
Cu alone and 100 µM Cu with MJ comparing all treatments,
but the difference was statistically not significant, whereas
carotenoids level was the highest.
The Fv/Fm ratio and Rfd index gained similar values in
all treatments used. The qP coefficient achieved the lowest
value for 100 µM Cu with MJ pre-incubated for 24 h of all
treatments examined. What is more, two other parameters -
NPQ and LNU were the highest for all treatments with Cu
pre-incubated with MJ in comparison with all treatments
tested. The %X parameter was not statistically different in
all treatments used, with exception for 50 µM Cu alone as
the lowest value.
Summarizing the results achieved on Phaseolus coccineus
plants in their early phase of growth, Cu produced a
marked inhibition in growth, leaves mass and caused enormous
permeability of membranes of all Cu-treated plants,
but only mild interference with the fluorescence-related parameters.
Moreover, MJ can partially act as a modifier of
copper toxicity in plants.