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Volume 47(1-4):115-117, 2003
Acta Biologica Szegediensis
http://www.sci.u-szeged.hu/ABS
SYMPOSIUM
1Institute of Pharmacognosy, Semmelweis University, Budapest, Hungary, 2 Chemical Institute, Chemical Research Center,
Hungarian Academy of Sciences, Budapest, Hungary, 3 Department of Genetics and Molecular Biology, University of
Szeged, Szeged, Hungary
Examination on antioxidant activity in the greater
celandine (Chelidonium majus L.) extracts by FRAP method+
Mária Then1*, Klára Szentmihályi2, Ágnes Sárközi1, Ilona Szöllôsi Varga3
ABSTRACT
Antioxidant activity in the alcoholic extracts (20 and 40%) of the greater
celandine (Chelidonium majus L.) herb was investigated by FRAP (ferric reducing and
antioxidant power) method. Since the antioxidant activity of the extracts greatly depends on
the quality of compounds, the phytochemical examination for alkaloid- and element content
were also examined. According to the results the antioxidant activity does not depend on the
alkaloid content of the drug during the vegetation period and on the alkaloid content of the
alcoholic extracts. It seems that the antioxidant activity of the extracts is also independent from
the transition metal element content. Acta Biol Szeged 47(1-4):115-117 (2003)
KEY WORDS
Chelidonium majus L.
alcoholic extract
FRAP method
alkaloid content
metal ion content
Accepted April 30, 2003
*Corresponding author. E-mail: thenm@drog.sote.hu
+In memory of Professsor Béla Matkovics
115
The greater celandine (
Chelidonium majus L
.) is not listed
in the Hungarian Pharmacopoeia in spite of the fact that the
drug is commercially available in the herb-trade. In folk
medicine the antiviral activity of the plant is attributed to its
alkaloids present in the freshly outflowing latex (Dickson
1996). The alkaloid components of the orange coloured latex
of the plant (chelidonine, chelerythrine, coptisine, sangui-
narine, berberine etc.; Then et al. 2000) also have a number
of beneficial effect,
e.g.
spasmolytic-, antiinflammatory-,
antimicrobial-, antiviral-, antifungal-, antitumor activity and
cytotoxic properties (Khayyal et al. 2001; Coon and Ernst
2002; Kokoska et al. 2002).
The antioxidant activity of the drug and extracts has not
examined yet. Therefore, the antioxidant activity was mea-
sured by FRAP method and evaluated on the bases of alka-
loid and element content. The the FRAP method means the
ferric reducing ability of plasma or plants (Benzie and Strain
1996, 1999). Ferric to ferrous ion reduction at low pH causes
a ferrous-tripyridyl-triazine complex which has absorbtion at
593 nm. FRAP values are obtained by comparing the absor-
bance changes at the given vawelength and how influence the
added plasma aliquots the FRAP values. Absorbance changes
are linear over a wide concentration range with antioxidant
mixture, including plasma or purified antioxidant mixture.
The known antioxidants are interact very much. The FRAP
assays are inexpensive, simple to prepare the reagents, the
results are highly reproducible and measurement takes no
long time.
Materials and Methods
Fresh aerial parts of greater celandine (
Chelidonium majus
L
.) were collected from the Botanical Garden of Budapest in
2002.
I. Extraction: The plant (5 g) was poured with alcoholic
water (100 ml, 20 and 40%, 60°C) and allowed to stand at
room temperature for 24 hours, then filtered.
II. Extraction for antioxidant activity of drug sample: The
plant (1.5 g) was poured with double distilled water (200 ml)
and allowed to stand at room temperature for 30 min, then
filtered.
For the determination of total alkaloid content of plant
and extracts, the reference method chosen was the
measurement of chelidonine content according to the
German Pharmacopoeia (DAB 10) as follows. The plant
(0.75g) or the solution (25 ml) was extracted with CH3COOH
(200 ml, 12%, g/v) by refluxing on a water bath for 30 min.
After cooling the solution was filtered into a volumetric flask
(250 ml). The acetic acid extract (30 ml) was made alkaline
(pH 8-9) with NH4OH (25%) then extracted with CHCl3
(3x30 ml) in a separatory funnel. The CHCl3 phase was
mixed and dried on anhydrous Na2SO4 and after filtration,
CHCl3 was evaporated under vacuum. The residue was
redissolved in CH3CH2OH (2.5 ml) and transferred into a
volumetric flask (25 ml). The trace residue was washed with
diluted H2SO4 (10%, 3x5 ml) and also transferred into the
volumetric flask, then the solution was diluted to 25 ml. A
mixture of this solution (5 ml) and chromotropic acid (5 ml)
was diluted with H2SO4 (98%) to 25 ml, then kept on a
boiling water bath (100°C) for 10 min. After cooling, the
absorption of the solution was measured at 570 nm against
116
Then et al.
the blank solution. The extinction coefficient in 1% solution
in a 1 cm vessel was 933 (100 ml g-1 cm-1).
The FRAP method for measuring the ferric reducing
ability of plasma (FRAP) or plants (Benzie and Strain 1996,
1999) is the following:
Reagents:
1. Acetate buffer, 300 mmol/L pH 3.6 (3.1 g sodium
acetate x3H2O and 16 ml acetic acid in 1000 ml buffer
solution).
2. 10 mmol/l 2,4,6-tripyridyl-s-triazine (TPTZ) in 40
mmol/l HCl.
3. 20 mmol/l FeCl3x6H2O in distilled water.
Element concentration of samples was determined by an
inductively coupled plasma atomic emission spectrometer
(ICP-AES). Type of instrument: Atom Scan 25 (Thermo
Jarrell Ash), a sequential emission spectrometer. Sample
handling: the samples (50 ml of evaporated extract ) were
digested with a mixture of HNO3 (5 ml) and H2O2 (3 ml) in
teflon vessels. After digestion the samples were diluted to 25
ml, from which the following elements were determined in
three parallel measurements: Ca, Cu, Fe, K, Mg, Mn, Na, P,
Zn.
Results and Discussion
Table 1. Alkaloid content (%, g/100 g) and antioxidant activity (µmol/l) of the greater celandine (Chelidonium majus L.) plant
herb during the vegetation period.
Harvested period Alkaloid content Antioxidant activity
(%) (µmol/L)
III. month 0.218 ± 0.023 569.5 ± 9.41
IV. month 0.196 ± 0.015 289.5 ± 018.5
VI. month 0.117 ± 0.008 422.2 ±32.1
Table 2. Alkaloid content (%, g/100 ml) and antioxidant activity (µmol/l) in the alcoholic extracts of the greater celandine
(Chelidonium majus L.).
Samples Alkaloid content (%) Antioxidant activity* (µmol/L)
20 % alcoholic extract 0.172 ± 0.008 90.6 ± 9.4
40 % alcoholic extract 0.380 ± 0.009 91.4 ± 15.2
*20 % and 40 % alcoholic-aqueous solution have no antioxidant activity.
Table 3. Element concentration (µg/ml) in the alcoholic extracts of the greater celandine (Chelidonium majus L.).
Elements 0 % alcoholic extrac 0 % alcoholic extract
Ca 10.94 ± 0.29 6.25 ± 0.20
Cu 0.273 ± 0.173 0.188 ± 0.074
Fe 0.217 ± 0.023 0.137 ± 0.029
K 52.36 ± 5.07 67.94 ± 0.17
Mg 17.01 ± 0.63 11.90 ± 0.12
Mn 0.141 ± 0.009 0.086 ± 0 008
Na 39.21 ± 0.80 23.50 ± 0.20
P 30.41 ± 0.27 19.83 ± 0.15
Zn 0.464 ± 0.006 0.346 ± 0.006
FRAP working solution: 25 ml acetate buffer (1), 2.5 ml
TPTZ solution and 2.5 ml FeCl3x6H2O solution.The work-
ing solution must be always freshly prepare.
Aqueous solution of known FeSO4x7H2O was used for
calibration.
Assay: Blank: FRAP reagent.
Sample: FRAP reagent 1,5 ml and sample solution 50 µl.
Monitoring up to 5 min at 593 nm, 1 cm light path and
37°C. Fe(II) standard solution tested in parallel. Calculation:
usig the calibration curve.
The drug samples was collected during the vegetation period
in different time to observe the changes of the antioxidant
activity with total alkaloid content. Total alkaloid content of
the samples was measured accordance with the German
Pharmacopoeia (DAB 10), and the antioxidant activity was
assayed by FRAP method (Table 1). According to the results
we stated that the alkaloid content of the drug changes during
the vegetation period and it seems that the antioxidant
activity does not depend on the alkaloid content of the drug.
117
Antioxidant activity of celandine
Alcoholic extracts (20 and 40%) of the greater celandine
contains different amount of alkaloids with almost the same
and very low antioxidant activity (Table 2). This result
confirms our previous measurements obtained during vege-
tation period. Alcoholic solutions (20 and 40%) do not have
an influence on the antioxidant activity.
The extracts contain elements as well dissolved in or
bound by organic compounds (Buzuk et al. 2001). Element
content of the extracts is very low (Table 3) and they do not
contain transition metal ions in higher concentration than
other alcoholic extracts (Szentmihályi and Then 2001;
Szentmihályi et al. 2001).
Though the greater celandine (aqueous extract) has
antioxidant activity, this value semms to be independent from
its alkaloid and element content.
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