Full Text (PDF) - Journal of Bioscience and Biotechnology

ISSN: 1314-6246
Popova et al.
J. BioSci. Biotech. 2014, SE/ONLINE: 17-21
Aneta T. Popova 1
Dasha S. Mihaylova 2
Iordanka N. Alexieva 1
The effect of freezing on the antioxidant activity
of Bulgarian Chrysanthemum balsamita
Authors’ addresses:
Department of Catering and Tourism,
University of Food Technologies,
Plovdiv, Bulgaria.
Department of Biotechnology,
University of Food Technologies,
Plovdiv, Bulgaria.
Aneta Popova
Department of Catering and Tourism,
University of Food Technologies,
Plovdiv, Bulgaria.
Tel.: +359 894856895
e-mail: [email protected]
The aim of the present paper was to investigate the antioxidant capacity and the
possible cell structure changes of frozen Chrysanthemum balsamita leaves. The
antioxidant activity of the studied aqueous extracts was estimated through ABTS,
DPPH, FRAP, and CUPRAC methods. The total phenolic content was assessed
as well. The results varied significantly before and after freezing of the plant
samples. The total phenolic content of the fresh leaves was established to be 0.59
mg GAE/g PW and 0.68 mg GAE/g PW of the frozen leaves.
The microscopic analysis of both fresh and frozen leaves showed changes in the
cellular structure which influenced the radical scavenging activity of the frozen
Chrysanthemum balsamita plant samples.
Key words: Chrysanthemum balsamita, costmary, antioxidant activity, cell
Aromatic, medicinal and spicy plants are an immense and
sustainable source of natural compounds with various
beneficial properties. They have been used since ancient ages
for various applications, particularly healing of diseases,
flavoring of foods and formulation of fragrances. Some of
these plants nowadays are grown commercially and are used
for the production of a variety of ingredients.
Costmary (Chrysanthemum balsamita L.) is a medicinal
plant, belonging to the Asteraceae family. It is a large
perennial plant of Asian origin with yellow flowers, grown in
Europe and Asia since the Middle Ages (Bylaitė et al., 2000).
This plant has a hairy stem, complete shiny leaves, highly
branched from the base and 70-120 cm height (Nickavar et
al., 2003; Mozaffarian, 2004; Hassanpouraghdam et al.,
2009). It has a characteristic odor due to its volatile oil
constituents. The main compounds (above 3%) are: carvone
(47.81%), α-thujone (12.56%), germacrene B (5.23%),
benzaldehyde (4.64%) ethylbenzene (3.96%) and germacrene
D (3.13%). Costmary has different phytochemicals such as;
oxygenated terpenes (73.5%), hydrocarbon terpenes (14.6%),
aromatic hydrocarbons (5.7%), aromatic oxygenated (4.7%)
and other components (1.5%) (Sapundzhieva et al., 2013). It
is also rich in phenylpropane derivatives, flavones,
sesquiterpene lactones, tannins and oligoelements (Gallori et
al., 2001). Costmary has been used as a spice for flavoring
different types of food, cakes, drinks, as well as in
confectionery since antiquity (Bylaitė et al., 2000; Gallori et
al., 2001). A herbal tonic tea of Costmary leaves traditionally
is consumed in various cultures (Abad et al., 2006).
Information on the effect of some technological factors on the
antioxidant activity of plant extracts is scares.
The objective of this study was to analyze the extracts of
fresh and frozen C. balsamita leaves for their radical
scavenging activity and the effect of the expected cellular
structure changes of the herb on its phenolic content and
antioxidant activity.
Materials and Methods
Plant material
Chrysanthemum balsamita L. plant samples were
collected in May 2013 from their natural habitat in Plovdiv
region, Bulgaria.
Extract preparation
The applied method was decoction – extracting by boiling
fresh/frozen/dried herbal and plant material. 2.5 g of the fresh
and frozen plant sample were boiled for 15 min in 50 ml of
distilled water. The resulting solution was being filtered
before analyzed.
Section “Cell & Molecular Biology”
Third Balkan Scientific Conference on Biology, Plovdiv, May 30 – June 1, 2014
ISSN: 1314-6246
Popova et al.
J. BioSci. Biotech. 2014, SE/ONLINE: 17-21
Determination of total polyhenolic content (TPhC)
A modified method (Kujala et al., 2000) was used for the
determination of the total polyphenolic content (TPhC). Each
extract was mixed with Folin-Ciocalteu reagent and 7.5%
Na2CO3. The mixture was vortexed and left for 5 min at
50°С. After incubation, the absorbance was measured at 765
nm and room temperature. Gallic acid was employed as a
calibration standard and the results were expressed as mg
gallic acid equivalents (mg GAE) per gram of plant weight.
mM HCl, and 20 mM iron (III) chloride solution in
proportions of 10:1:1 (v/v), respectively. The FRAP reagent
was prepared fresh daily and was warmed to 37°C in a water
bath prior to use. One hundred and fifty microliters of plant
extracts were allowed to react with 2850 µl of the FRAP
reagent solution for 4 min at 37°C and the absorbance of the
reaction mixture was recorded at 593 nm. The results were
expressed as µM TE/g PW.
CUPRAC assay
Antioxidant activity was described as having activity
against the stable form of the synthetic product DPPH • (2.2diphenyl-1-pikrylhydrazil) by the method of Brand-Williams
et al. (1995) with slight modifications. A freshly prepared
14.10-5 M solution of DPPH• (in methanol) was mixed with
the sample in a ratio of 2:0.5. The unit of Trolox equivalent
antioxidant capacity (TEAC) defined the concentration of
Trolox having equivalent AOA expressed as μM TE/g PW.
The CUPRAC assay was carried out according to the
procedure of Apak and coworkers (Apak et al., 2004) with
modifications. To a test tube were added 1 ml of CuCl2
solution (1.0×10−2 M), 1 ml of neocuproine methanolic
solution (7.5×10−3 M), and 1 ml NH4Ac buffer solution (pH
7.0), and mixed; 0.1 ml of herbal extract (sample) followed
by 1 ml of water were added (total volume = 4.1 ml), and
mixed well. Absorbance against a reagent blank was
measured at 450 nm after 30 min. Trolox was used as
standard and total antioxidant capacity of herbal extracts was
measured as μM TE/g PW.
ABTS•+ radical scavenging assay
Microscopic analysis
Determination of antioxidant activity
DPPH • radical scavenging assay
The radicals scavenging activity of the ultrasound extract
against radical cation (ABTS•+) was estimated according to a
previously reported procedure with some modifications (Re
et al., 1999). ABTS•+ was produced by reacting 7 mM of
ABTS•+ solution with 2.45 mM of potassium persulphate, and
the mixture was kept in the dark at room temperature for 1216 h. At the moment of use, the ABTS•+ solution was diluted
with ethanol to an absorbance of 0.7 ± 0.02 at 734 nm and
equilibrated at 30°C. One ml of ABTS•+ solution was added
to each sample (0.01 ml) was vigorously mixed. After
reacting at 30°C temperature for 6 min, the absorbance at 734
nm was measured. The TEAC value was defined as the
concentration of Trolox having equivalent antioxidant
activity expressed as µM TE per gram plant weight (µM
TE/g PW).
Ferric-reducing antioxidant power assay (FRAP)
The FRAP assay was carried out according to the
procedure of Benzie and Strain (1996) with slight
modification. FRAP assay measures the change in
absorbance at 593 nm owing to the formation of a blue
colored Fe (II)-tripyridyltriazine compound from colorless
oxidized Fe (III) form by the action of electron donating
antioxidants. Briefly, the FRAP reagent was prepared from
300 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40
Thin sections of the plant leaf were prepared by handcutting at a 40-45° angle of the cutting knife. The section
should be as thin as possible. It was mounted on a
microscope slide using tweezers. The thinner the cut the less
the section curled (liquid may be applied for retrieving a
better sample). A cover glass was pressed carefully either by
hand or with tweezers (no air should be left between the slide
and the cover glass). Photomicrographs of x400
magnifications were taken with Celestron Deluxe LCD
Digital microscope and analyzed.
Statistical analysis
All measurements were carried out in triplicates. The
results were expressed as mean ± SD and statistically
analyzed using MS-Excel software.
Results and Discussion
Total phenolics
The total phenolic contents in the examined plant extracts
using the Folin-Ciocalteu’s reagent was expressed in terms of
gallic acid equivalents (Table 1). Freezing seams to affect
TPhC of the examined extracts showing values of the fresh
leaves 0.59 mg GAE/g PW and of the frozen samples - 0.68
mg GAE/g PW.
Section “Cell & Molecular Biology”
Third Balkan Scientific Conference on Biology, Plovdiv, May 30 – June 1, 2014
ISSN: 1314-6246
Popova et al.
J. BioSci. Biotech. 2014, SE/ONLINE: 17-21
Table 1. Total phenol content (mg GAE/g PW) and in vitro antioxidant activity (μM TE/g PW) of Chrysanthemum balsamita
aqueous extracts.
Plant sample decoction
Fresh leaves
Frozen leaves
0.59 ± 0.04
0.68 ± 0.01
10.68 ± 0.25
12.01 ± 0.07
3.55 ± 0.01
3.02 ± 0.01
7.35 ± 0.12
8.79 ± 0.28
5.93 ± 0.07
6.22 ± 0.09
Microscopic analysis
Antioxidant activity
The antioxidant activity of aqueous extracts of
Chrysanthemum balsamita was assessed with the use of four
different assays: DPPH• and ABTS•+, FRAP and CUPRAC.
These assays are widely used for determination of total AOA
in many foods such as fruits, vegetables and spices (Prior et
al., 2005; Ciz et al., 2010).
In order to investigate the antioxidant activity of the plant
extracts, experiments with two stable radicals DPPH• and
ABTS•+ were conducted. A higher TEAC value indicates
stronger antioxidant activity. The results represented in Table
1 revealed that all extracts possessed free radical-scavenging
activity but at different levels.
Significant ABTS free radical scavenging activity was
evident in the 15 min decoction of frozen leaves (12.01 ±
0.07 µM TE/g PW). The antioxidant potential of C.
balsamita extracts was estimated from their ability to reduce
TPRZ-Fe (III) complex to TPTZ-Fe (II) as well. In
accordance with the ABTS assay and the total polyphenolics,
the highest value in the FRAP method was obtained in the
decoction extract of frozen costmary leaves - 8.79 ± 0.28 µM
TE/g PW. The cupric ion (Cu2+) reducing ability of the
extracts of C. balsamita leaves was also evaluated. Among
the two extracts, the decoction of frozen leaves showed the
higher CUPRAC value – 6.22 ± 0.09 µM TE/g PW. The
results of this assay correspond well to the already mentioned
results pursuant to the ABTS, FRAP and TPC methods.
Contrary to all the other conducted studies the DPPH assay
confirmed the higher values established by the decoction of
fresh leaves (3.55 ± 0,01 µM TE/g PW). ). In comparison, the
TEACDPPH value for the frozen leaves was 3.02 ± 0.01 µM
TE/g PW. This is probably due to the different mechanism of
contribution of each individual component to the total radical
scavenging activity of the studied samples.
The surface of the fresh leaves was characterized by a
mosaic structure (Figure 1). Under the microscope the outer
edges had irregular shape and were transparent. Cell walls
were distinguished in the fresh leaves (Figure 2). Fine
transparent hairs can be seen in the middle green part of the
fresh and frozen leaves (Figure 3, Figure 4). In the examined
material of the frozen leaves structure changes were visible
(Figure 5). Changes were spotted in the stems as well (Figure
6, Figure. 7). Further analysis would have to clear the
mechanism and how freezing (-18°C) structural changes
influence the radical scavenging ability of C. balsamita
leaves. Probably changes occurred at the cellular level which
altered the structure of the cells and the extraction of
chemical substances. It was assumed that by freezing water
forms crystals that mechanically damaged cell walls. Protein
denaturation and aggregation, inactivation of enzymes in
chloroplasts and mitochondria and increased fluidity of
membrane lipids is likely to affect membrane permeability.
Probably ruptured cell walls have led to a more complete
extraction of the cell consisted substances.
Figure 1. Microscopic image of surface structure of fresh
costmary leaf.
Section “Cell & Molecular Biology”
Third Balkan Scientific Conference on Biology, Plovdiv, May 30 – June 1, 2014
ISSN: 1314-6246
Popova et al.
J. BioSci. Biotech. 2014, SE/ONLINE: 17-21
Figure 2. Microscopic image of fresh costmary leaf.
Figure 5. Microscopic image of frozen costmary leaf.
Figure 3. Microscopic image of middle part of fresh
costmary leaf.
Figure 6. Microscopic image of fresh costmary stem.
Figure 4. Microscopic image of middle part of frozen
costmary leaf.
Figure 7. Microscopic image of frozen costmary stem.
Section “Cell & Molecular Biology”
Third Balkan Scientific Conference on Biology, Plovdiv, May 30 – June 1, 2014
ISSN: 1314-6246
Popova et al.
J. BioSci. Biotech. 2014, SE/ONLINE: 17-21
Costmary shows a great potential as an ingredient of
foods containing compounds with antioxidant activity.
Freezing fresh plants is a common way of storing used in the
food technology. Being stored in a freezer the costmary
leaves are very likely to experience cell changes leading to a
higher radical scavenging capacity.
The research leading to these results has received funding
from the European Community's Seventh Framework
Programme (FP7/2007-2013) under grant agreement n.
227118, project BaSeFood.
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Section “Cell & Molecular Biology”
Third Balkan Scientific Conference on Biology, Plovdiv, May 30 – June 1, 2014