Vaccinium myrtillus L. (bilberry) is a plant native to Lithuanian forests. The high concentration of anthocyanins (Burdulis et al. 2009) gives to bilberry important antioxidant properties (Takikawa et al. 2010), making this plant very demanded in market. Many studies have been conducted to analyse the actual beneficial properties of bilberry, but most of these studies are related to the quantity of anthocyanins present in the fruits. There are only few studies focused on the leaves, twigs and roots of bilberry. This research work intends to analyse and compare the quantity of phenolic compounds, flavonoids and radical scavenging activity present in the aerial parts and roots of Lithuanian bilberry grown in two different forest conditions and sampled in three different vegetative periods. The two different areas of the forest had a clear cutting in two different periods. The area that will be named “5b” had a clear cutting in early 2017; the area that will be named “7b” had a clear cutting in early 2015. All the samples have been taken during the year 2017, but in different vegetation periods: early May, late May and early August. The aim of this research work is to analyse the influence that different forest areas and different vegetation periods may have in the amounts of phenolic compounds, flavonoids and radical scavenging activity of the samples extract. In the first part of this work, it will be given a botanical and general description of bilberry, following phytochemical and pharmacological properties. In the second part, it will be given information about the material and methods used for the analysis of the samples extract. In the last part of this work, all the data obtained will be reported and discussed in detail.

1. VYTAUTAS MAGNUS UNIVERSITY
FACULTY OF NATURAL SCIENCES
APPLIED BIOTECHNOLOGY
COMPARISON OF PHENOLIC COMPOUNDS, FLAVONOIDS AND
RADICAL SCAVENGING ACTIVITY IN VACCINIUM MYRTILLUS L.
GROWN IN DIFFERENT FOREST CONDITIONS
AND SAMPLED IN DIFFERENT VEGETATION PERIODS
RESEARCH PROJECT
Student:
Salvatore Paradiso
Supervisor:
Nicola Tiso
Kaunas, 2018

2. 1
Contents
1. Introduction 2
2. Characteristics
2.1 Botanical and general description 2
2.2 Phytochemical characteristics and pharmacological properties 3
3. Material and methods
3.1 Raw material 4
3.2 Determination of total content of phenolic compounds 5
3.3 Determination of total content of flavonoids 6
3.4 Determination of radical scavenging activity 7
4. Results and discussion
4.1 Humidity test 7
4.2 Phenolic compounds concentration 8
4.3 Flavonoids concentration 12
4.4 Radical scavenging activity 15
5. Conclusions 18
Acknowledgment 19
References 20

3. 2
1. Introduction
Vaccinium myrtillus L. (bilberry) is a plant native to Lithuanian forests. The high
concentration of anthocyanins (Burdulis et al. 2009) gives to bilberry important antioxidant properties
(Takikawa et al. 2010), making this plant very demanded in market. Many studies have been
conducted to analyse the actual beneficial properties of bilberry, but most of these studies are related
to the quantity of anthocyanins present in the fruits. There are only few studies focused on the leaves,
twigs and roots of bilberry. This research work intends to analyse and compare the quantity of
phenolic compounds, flavonoids and radical scavenging activity present in the aerial parts and roots
of Lithuanian bilberry grown in two different forest conditions and sampled in three different
vegetative periods. The two different areas of the forest had a clear cutting in two different periods.
The area that will be named “5b” had a clear cutting in early 2017; the area that will be named “7b”
had a clear cutting in early 2015. All the samples have been taken during the year 2017, but in
different vegetation periods: early May, late May and early August. The aim of this research work is
to analyse the influence that different forest areas and different vegetation periods may have in the
amounts of phenolic compounds, flavonoids and radical scavenging activity of the samples extract.
In the first part of this work, it will be given a botanical and general description of bilberry, following
phytochemical and pharmacological properties. In the second part, it will be given information about
the material and methods used for the analysis of the samples extract. In the last part of this work, all
the data obtained will be reported and discussed in detail.
2. Characteristics
2.1 Botanical and general description
European blueberry, or bilberry (Vaccinium myrtillus L.) is a plant of the Ericaceae family
that belongs to the genus Vaccinium, which includes around 200 species widespread across the world
(Jaakola 2003). Bilberry is a deciduous woody dwarf shrub plant, native to Europe, that can reach 40
cm in high; the leaf has elliptic-oval shape with a dentate margin, light green, long about 2-3 cm; the
flowers are hermaphrodite, whitish or yellow-greenish; the fruit is a berry of around 5-8 mm, blue,
black or purple in colour (Ferrari and Medici 2008). The European blueberry is often confused with
the American blueberry. The difference between the two berries is in the pulp: the European blueberry
has a purple pulp; the American blueberry has a cream or white pulp (Valentovà et al. 2007).
Bilberry is typical and abundant in the northern hemisphere especially in spruce and pine
forests, with better-drained acid soils of medium fertility, and it is precious for many birds and

4. 3
mammals, which it constitutes an important food resource (Nestby et al. 2011). In North Europe,
bilberry is one of the most important wild berries and its good taste and aroma makes it highly
demanded in the market, although it is still not very cultivated but harvested in forest fields (Uleberg
et al. 2012). Nevertheless, bilberry has been used by humans as food for centuries due to its high
nutritive value, and as medical plant since Middle Ages (but it became widely known to herbalists
only in the 16th
century) (Valentovà et al. 2007).
Leaves and twigs of bilberry are often used as herbal tea. A research conducted within the
EU/EEA Member States, has shown the presence of dry bilberry in the market for more than 30 years
as single active ingredient. This research has shown that on the German market there are 115 bilberry
herbal teas, used mostly to heal conditions such as unspecific acute diarrhoea and mild inflammation
of the oropharyngeal mucosa; on the Italian market since at least 1984, a methanolic bilberry extract
has been present, prepared from fresh fruit and containing 25% of anthocyanidins and sold in capsules
or as a granulate (European Medicine Agency 2015).
2.2 Phytochemical characteristics and pharmacological properties
A great variety of phenolic compounds, very often situated in the surface layer, can be found
in plant tissues where they conduct their main function, to protect the plant against biotic and abiotic
factors (Nohynek et al. 2006). Phenolic compounds are classified into four main groups: flavonoids,
phenolic acids, lignans, and polymeric tannins. The phenolic content in vegetables however is not
stable and after a long-term storage in the freezer it tends to decrease (Nohynek et al. 2006). The
environmental conditions, the vegetative phase and the different parts of a plant, affect the quantity
and the quality of phenolic compounds present in the samples (Bujor et al. 2016).
The Vaccinium genus is well known for including plant species noted for their high content
of anthocyanins (water soluble pigments belonging to the flavonoid family), that are involved in many
biological activities and that may affect positively the health (Burdulis et al. 2009). The high
concentration of anthocyanins gives to bilberry high antioxidant properties (Takikawa et al. 2010).
Antioxidant are very important for living systems. High levels of free radicals can lead to the
oxidation of biomolecules and, as consequence, to the damage or death of cells, to tissue damages or
to various disease such as cancer. Antioxidant can inhibit the effect of oxidants by donating hydrogen
atoms or by chelating metals (Sen et al. 2000). For this reason, the researches on bilberries high
antioxidant capacity have been of interest among consumers, who want a better understanding on
how this berry can contribute to improve the nutritional quality of their diet (Poiana et al. 2012).
Furthermore, many studies have reported that the complex mixture of bilberry’s anthocyanins may

5. 4
be helpful to fortify blood vessel walls, to increase the flexibility of the capillaries, to improve blood
flow and to maintain a good blood circulation (Poiana et al. 2012).
A study conducted by Suzuki et al. has shown that the anthocyanidin enriched bilberry extracts
may be used as a complementary treatment for obese patients with metabolic syndrome, due to their
strong efficiency to inhibit the adipocyte differentiation via the insulin pathway (Suzuki et al. 2011).
Another study conducted by Güder et al. has shown that the extracts of Vaccinium myrtillus L., due
to the presence of anthocyanins, inhibit α-amylase and α-glucosidase enzymes, making bilberry a
possible natural therapeutic food for diabetes (Güder et al. 2015). The same study of Güder et al. has
also confirmed that anthocyanins in Vaccinium myrtillus L. extracts have strong antioxidant
properties.
A study conducted by Yamaki and Kobori has shown that the ethanol extract of bilberry was
found to be the most effective against the growth of HL60 human leukaemia cells, and against
HTC116 human colon carcinoma cells in vitro. This study was conducted with 10 different edible
berries. The extract of bilberry contained the largest amounts of phenolic compounds (including the
largest amounts of anthocyanins) and it had the greatest radical scavenging activity (Yamaki and
Kobori 2003).
3. Material and methods
3.1 Raw material
The samples of Vaccinium myrtillus L. were taken from two different areas of the same forest
with different cutting conditions, situated near Kaunas (Lithuania). The area named “5b” had a clear
cutting in 2017, and the samples were taken after the cutting; the area named “7b” had a clear cutting
in 2015, and the samples were taken two years after the cutting (2017). The average annual
temperature in Lithuania is between +6°C and –7°C. In winter the average minimal monthly
temperature is about –24°C to –26°C. The annual precipitation is 500-750 mm. The coldest month is
January, the warmest is July and the rainiest are July and August (based on long-time observations).
Of the total annual precipitation, the 60% falls during the warm period of the year (Bukantis 1994).
The samples were taken during three different vegetation periods: early May, late May and
early August (2017). From each sample were taken the aerial parts of the plant and the roots. In total
were analysed 6 samples from the “5b” area and 6 samples from the “7b” are. The following tabs
show the list of the samples analysed (Tab.1 and Tab.2):

6. 5
ID Pick up date Part of the plant
1 05-05-2017 Aerial parts
2 05-05-2017 Roots
3 30-05-2017 Aerial parts
4 30-05-2017 Roots
5 01-08-2017 Aerial parts
6 01-08-2017 Roots
ID Pick up date Part of the plant
7 05-05-2017 Aerial parts
8 05-05-2017 Roots
9 24-05-2017 Aerial parts
10 24-05-2017 Roots
11 01-08-2017 Aerial parts
12 01-08-2017 Roots
The samples of Vaccinium myrtillus L. were firstly reduced to a fine powder with a blender.
From the powder obtained it was conducted a humidity test with a PMB 53 (Adam Equipment, USA)
apparatus. The results of this tests will be used for the determination in milligrams of phenolic
compounds, flavonoids and radical scavenging activity in 1 g of dry material of bilberry. The samples
extract were prepared with 0.5 g of fine bilberry powder, added in a solution of 20 ml 75% methanol
and bi-distilled water. The samples were left in a shaker for 24h and after were filtered with a filtering
paper and stored in the refrigerator at 5°C.
3.2 Determination of total content of phenolic compounds
The total amount of phenolic compounds was evaluated using the method described by
Mikašauskaitė et al. (2013). The samples extract were firstly diluted 10 times with a solution of 75%
methanol and bi-distilled water. A stock solution of 4% Na2CO3 (sodium carbonate) in bi-distilled
water was prepared and stored in the refrigerator at 5°C. For standard compound was used rutin (0.01
– 1.00 mg/ml) and the calibration curve was built at six concentrations. From each concentration of
Tab.1 – “5b” area (clear cutting 2017) samples.
Tab.2 – “7b” area (clear cutting 2015) samples.

7. 6
rutin were measured three absorptions and the average of them was used for building the calibration
curve
From each diluted sample extract were taken 50 µl and added to 1500 µl of stock solution.
Each vial was inverted two times and well mixed with a vortex. In each vial were added 50 µl of
Folin – Ciocalteu reagent (2N) and, after incubating the vials for 30 minutes at room temperature, the
absorbance was measured at 760 nm. For calibrating the spectrophotometer was used a Blank of 50
µl of methanol 75% and 1500 µl of stock solution; 50 µl of Folin – Ciocalteu reagent (2N) were added
in the Blank and, after incubating the vial for 30 minutes at room temperature, the absorbance was
measured at 760 nm. For each sample the absorbance was measured three times and the average of
the results was used for the determination of the concentration of phenolic compounds. All the
measurements were conducted with a Milton Roy Spectronic 1201 (Milton Roy, USA) apparatus.
3.3 Determination of total content of flavonoids
The total flavonoid content was determined using the method described by Kaškonienė et al.
(2009). For the determination of the total content of flavonoids the samples were not diluted. The
stock solution was prepared with 60 ml of methanol (100%), 3 ml of acetic acid (33%), 12 ml of
hexamethylenetetramine (5%), 9 ml of aluminium chloride (10%) and 60 ml of bi-distilled water. The
stock solution was left to incubate in the refrigerator (5°C) for a day. For standard compound was
used rutin (0.01 – 1.00 mg/ml) and the calibration curve was built at six concentrations. From each
concentration of rutin were measured three absorptions and the average of them was used for building
the calibration curve.
From each samples extract were taken 40 µl and added to 960 µl of stock solution (5°C), each
vial was inverted two times and well mixed with a vortex. After 30 minutes of incubation the
absorbance was measured at 407 nm. For calibrating the spectrophotometer was used a Blank of 40
µl of methanol (75%) and 960 µl of stock solution, and, after incubating the vials for 30 minutes at
room temperature, the absorbance was measured at 407 nm. For each sample the absorbance was
measured three times and the average of the results was used for the determination of the
concentration of flavonoids. All the measurements were conducted with a Milton Roy Spectronic
1201 (Milton Roy, USA) apparatus.

8. 7
3.4 Determination of radical scavenging activity
The Radical scavenging activity was determined according to the modified method described
by Kaškonienė et al. (2009). The samples extract were firstly diluted 10 times with a solution of 75%
methanol and bi-distilled water. The buffer was prepared with 250 ml of 100 mM acetate buffer
solution with pH 5.5. To get the required pH it was used titrate with ca. 33% acetic acid. The radical
solution was prepared in a dark room with 100 mM DPPH radical solution (10 mg were dissolved in
125 ml acetonitrile and later were added 125 ml of methanol 100%). The buffer and the radical
solution were well mixed together in a light-proof bottle. The absorbance of the radical solution was
measured at 515 nm giving a result of 0.591. The stock solution was left in the dark for 15 minutes
before starting the analysis. For building the calibration curve it was used standard compound rutin
(0.05 – 0.25 mg/ml) at four different concentrations.
From each samples extract were taken 38.5 µl and added to 1500 µl of radical solution, each
vial was inverted two times and well mixed with a vortex. After 15 minutes of incubation the
absorbance was measured at 515 nm. The spectrophotometer was calibrated with a Blank sample
made with methanol (75%). All the measurements were conducted with a Milton Roy Spectronic
1201 (Milton Roy, USA) apparatus.
4. Result and discussion
4.1 Humidity test
The results of the humidity tests will be used for the determination in milligrams of phenolic
compounds, flavonoids and radical scavenging activity in 1 gram of dry material of bilberry. The
Tab.3 and Tab.4 show the results of the humidity test of the “5b” area samples and of the “7b” area
samples respectively:
ID Sample Humidity test %
1 05-05-2017 - Aerial parts 49.22
2 05-05-2017 - Roots 38.46
3 30-05-2017 - Aerial parts 49.98
4 30-05-2017 - Roots 37.85
5 01-08-2017 - Aerial parts 45.54
6 01-08-2017 - Roots 34.71
Tab.3 – “5b” area (clear cutting 2017) samples.
humidity test (%).

9. 8
ID Sample Humidity test %
7 05-05-2017 - Aerial parts 49.53
8 05-05-2017 - Roots 38.25
9 24-05-2017 - Aerial parts 52
10 24-05-2017 - Roots 35.96
11 01-08-2017 - Aerial parts 41.7
12 01-08-2017 - Roots 31.93
The results show that there is a higher percentage of humidity in the aerial parts of the plants
samples extract than in the roots. The results of the humidity test show also that there is no difference
in humidity percentage between the samples of the “5a” area and the “7b” area. From both the areas,
the sample taken during the late and early May vegetation period share a higher percentage of
humidity than the samples taken in early August.
4.2 Phenolic compounds concentration
The Tab.5 shows the results of rutin absorbance at six different concentrations.
Concentration
(mg/ml)
Absorbance 1 Absorbance 2 Absorbance 3 Average
1 1.031 1.069 1.059 1.053
0.8 0.753 0.710 0.730 0.731
0.6 0.615 0.605 0.612 0.611
0.4 0.446 0.464 0.456 0.455
0.2 0.278 0.272 0.265 0.272
0.1 0.097 0.094 0.104 0.098
The results of rutin absorbance at the six different concentrations were used for building the
calibration curve (Fig.1).
Tab.4 – “7b” area (clear cutting 2015) samples.
midity test (%).
Tab.5 – Rutin absorbance at 6 different concentrations.

10. 9
The Tab.6 and Tab.7 show the results of the average absorbance (760 nm) of the samples
extracts. From the average absorbance of the samples extract and from the equation of the calibration
curve (Fig.1) it was possible to estimate the concentration of phenolic compounds of the samples
from the different vegetation phases and from the two different areas of growth (Tab.6 and Tab.7).
ID Sample
Average
Absorbance
Dilution Concentration
1 05-05-2017 - Aerial parts 0.347 x 10 3.39
2 05-05-2017 - Roots 0.148 x 10 1.43
3 30-05-2017 - Aerial parts 0.317 x 10 3.16
4 30-05-2017 - Roots 0.108 x 10 1.06
5 01-08-2017 - Aerial parts 0.337 x 10 3.33
6 01-08-2017 - Roots 0.231 x 10 2.23
Fig.2 – Calibration curve
y = 1,0183x
R² = 0,9738
0,00
0,20
0,40
0,60
0,80
1,00
1,20
0 0,2 0,4 0,6 0,8 1 1,2
Absorbance(AU)
Concentration (mg/ml)
Fig.1 – Phenolic compounds calibration curve
Tab.6 – “5b” area (clear cutting 2017) samples extracts average phenolic compounds absorbance
and concentration.

11. 10
ID Sample
Average
Absorbance
Dilution Concentration
7 05-05-2017 - Aerial parts 0.169 x 10 3.43
8 05-05-2017 - Roots 0.107 x 10 1.09
9 24-05-2017 - Aerial parts 0.351 x 10 3.45
10 24-05-2017 - Roots 0.144 x 10 1.39
11 01-08-2017 - Aerial parts 0.421 x 10 4.19
12 01-08-2017 - Roots 0.131 x 10 1.29
From the results of the concentrations and from the results of the humidity tests it was
calculated the amount in milligram of phenolic compound in 1 g of dry material. The Fig.2 shows the
results of the “5b” area samples extract; the Fig.3 shows the results of the “7b” area samples extracts.
Fig.2 – “5b” area samples extract; milligrams of phenolic compounds
in 1 g of dry material. Rutin equivalent mg/1 g dry material.
Tab.7 – “7b” area (clear cutting 2015) samples extracts average phenolic compounds absorbance
and concentration.
0,00
50,00
100,00
150,00
200,00
250,00
300,00
1 2 3 4 5 6

12. 11
From the previous figures (Fig.2 and Fig.3) it is possible to see a difference in the
concentration of phenolic compounds (mg) present in a gram of dry material between the aerial parts
and the roots of the samples analysed. The areal parts of the samples are richer in phenolic compounds
in all the analysed samples. The results show also that there is not a real difference between the
quantity of phenolic compounds and the two different forest areas (5b and 7b) from which the samples
were taken. The concentration of phenolic compounds of the aerial parts from the samples of the “7b”
area is slight higher than the one from the samples of the “5b” area. The difference is only about
30mg/g dry material of the average results. This means that the different year on which the clear
cutting in the forest was made, did not influence the amounts of phenolic compounds in the analysed
samples. The different vegetation periods (beginning of May, late May and early August) of the
analysed samples did not also influence the quantity of phenolic compounds, therefore the results
between the samples are very similar. It is possible to observe higher amounts of phenolic compounds
only in the roots of the sample n°6 from the “5b” area (early August). This sample has an amount of
phenolic compounds of 137 mg/g of dry material (rutin equivalent) and it has a double concentration
of phenolic compounds compared to the sample n°4 of the same area (68mg/g dry material).
Fig.3 – “7b” area samples extract; milligrams of phenolic compounds
in 1 g of dry material. Rutin equivalent mg/1 g dry material.
0,00
50,00
100,00
150,00
200,00
250,00
300,00
350,00
7,00 8,00 9,00 10,00 11,00 12,00

13. 12
4.3 Flavonoids concentration
The Tab.8 shows the results of rutin absorbance at six different concentration.
Concentration
(mg/ml)
Absorbance 1 Absorbance 2 Absorbance 3 Average
1 1.021 1.025 1.017 1.021
0.8 0.752 0.741 0.735 0.743
0.6 0.570 0.560 0.564 0.565
0.4 0.424 0.457 0.440 0.440
0.2 0.235 0.244 0.241 0.240
0.1 0.115 0.131 0.119 0.122
The results of rutin absorbance at the six different concentrations were used for building the
calibration curve (Fig.4).
The Tab.9 and Tab.10 show the results of the average absorbance (407 nm) of the samples
extracts. From the average absorbance of the samples extract and from the equation of the calibration
curve (Fig.4) it was possible to calculate the concentration of flavonoids of the samples extract from
the different vegetation phases and from the two different areas of growth (Tab.9 and Tab.10).
Fig.2 – Calibration curve
Tab.8 – Rutin absorbance at 6 concentrations.
y = 0,9911x
R² = 0,9844
0,000
0,200
0,400
0,600
0,800
1,000
1,200
0 0,2 0,4 0,6 0,8 1 1,2
Absorbance(AU)
Concentration (mg/ml)
Fig.4 – Flavonoids calibration curve

14. 13
ID Sample
Average
Absorbance
Dilution Concentration
1 05-05-2017 - Aerial parts 0.480 No 0.484
2 05-05-2017 - Roots 0 No 0
3 30-05-2017 - Aerial parts 0.356 No 0.359
4 30-05-2017 - Roots 0 No 0
5 01-08-2017 - Aerial parts 0.453 No 0.457
6 01-08-2017 - Roots 0 No 0
ID Sample
Average
Absorbance
Dilution
Real
concentration
7 05-05-2017 - Aerial parts 0.428 No 0.432
8 05-05-2017 - Roots 0 No 0
9 24-05-2017 - Aerial parts 0.420 No 0.424
10 24-05-2017 - Roots 0 No 0
11 01-08-2017 - Aerial parts 0.595 No 0.600
12 01-08-2017 - Roots 0 No 0.000
From the results of the concentrations and from the results of the humidity tests it was
calculated the amount in milligrams of flavonoids in 1 g of dry material. The Fig.5 shows the results
of the “5b” area samples extract; the Fig.6 shows the results of the “7b” area samples extracts.
Tab.9 – “5b” area (clear cutting 2017) samples extracts average flavonoids absorbance and
concentration
Tab.10 – “7b” area (clear cutting 2015) samples extracts average flavonoids absorbance and
concentration

15. 14
The results (Fig.5 and Fig.6) show that there is an absence of flavonoids content in the roots
of all the analysed samples from both the two different areas of the forest and from all the different
vegetation periods. The aerial parts of the analysed samples have a similar flavonoids content. There
is not a high difference in the content of flavonoids between the samples of the two different forest
areas and from the different vegetation periods. The sample n°11 from the “7b” area (early August)
has the higher amount of flavonoids (41.17 mg/g dry material); the sample 3 from the “5b” areas (late
May) has the lower amount of flavonoids (28.72 mg/g dry material). The other samples share very
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
45,00
1 2 3 4 5 6
Fig.5 – “5b” area samples extract; milligrams of flavonoids in 1 g of
dry material. Rutin equivalent mg/1 g dry material.
0,00
5,00
10,00
15,00
20,00
25,00
30,00
35,00
40,00
45,00
50,00
7 8 9 10 11 12
Fig.6 – “7b” area samples extract; milligrams of flavonoids in 1 g of
dry material. Rutin equivalent mg/1 g dry material.

16. 15
similar results. The results show that the different area from which the samples were taken and the
different vegetation periods, did not influence the concentration of flavonoids of the samples extract.
4.4 Radical scavenging activity
The Tab.11 shows the results of rutin absorbance at four different concentrations.
Concentration
(mg/ml)
Absorbance 1 Absorbance 2 Absorbance 3 Average
0.25 0.062 0.078 0.071 0.070
0.2 0.159 0.155 0.183 0.166
0.1 0.320 0.323 0.295 0.313
0.05 0.16 0.444 0.444 0.435
The results of rutin absorbance at the four different concentrations were used for building the
calibration curve (Fig.7).
The Tab.12 and Tab.13 show the results of the average absorbance (515 nm) of the samples
extracts. From the average absorbance of the samples extract and from the equation of the calibration
curve (Fig.7) it was possible to calculate the concentration of radical scavenging activity of the
Tab.11 – Rutin absorbance at 4 concentrations.
y = -1,7513x + 0,5085
R² = 0,9913
0,000
0,050
0,100
0,150
0,200
0,250
0,300
0,350
0,400
0,450
0,500
0 0,05 0,1 0,15 0,2 0,25 0,3
Absorbance(AU)
Concentration (mg/ml)
Fig.7 – Radical scavenging activity calibration curve.

17. 16
samples from the different vegetation phases and from the two different areas of growth (Tab.12 and
Tab.13).
ID Sample
Average
Absorbance
Dilution Concentration
1 05-05-2017 - Aerial parts 0.135 x 10 2.133
2 05-05-2017 - Roots 0.337 x 10 0.977
3 30-05-2017 - Aerial parts 0.177 x 10 1.891
4 30-05-2017 - Roots 0.377 x 10 0.751
5 01-08-2017 - Aerial parts 0.218 x 10 1.657
6 01-08-2017 - Roots 0.308 x 10 1.145
ID Sample
Average
Absorbance
Dilution Concentration
7 05-05-2017 - Aerial parts 0.210 x 10 1.703
8 05-05-2017 - Roots 0.393 x 10 0.658
9 24-05-2017 - Aerial parts 0.204 x 10 1.739
10 24-05-2017 - Roots 0.329 x 10 1.025
11 01-08-2017 - Aerial parts 0.137 x 10 2.123
12 01-08-2017 - Roots 0.329 x 10 1.027
From the results of the concentrations and from the results of the humidity tests it was
calculated the amount in milligrams of radical scavenging activity in 1 g of dry material. The Fig.8
shows the results of the “5b” area samples extract; the Fig.9 shows the results of the “7b” area samples
extracts.
Tab.12 – “5b” area (clear cutting 2017) samples extract average radical scavenging activity
absorbance and concentration
Tab.13 – “7b” area (clear cutting 2017) samples extract average radical scavenging activity
absorbance and concentration

18. 17
The results (Fig.8 and Fig.9) show that the radical scavenging activity is higher in the aerial
parts samples extract than in the roots samples extract. In the aerial parts samples extract from the
“5b” area, the radical scavenging activity seems to decrease with the progress of the vegetation
periods. This does not happen for the roots samples extract of the same area, that share similar results.
The radical scavenging activity in the “7b” area is similar between the aerial parts samples extract
0,00
20,00
40,00
60,00
80,00
100,00
120,00
140,00
160,00
180,00
200,00
1 2 3 4 5 6
Fig.8 – “5b” area samples extract; milligrams of radical scavenging
activity in 1 g of dry material. Rutin equivalent mg/1 g dry material.
0,00
20,00
40,00
60,00
80,00
100,00
120,00
140,00
160,00
180,00
1 2 3 4 5 6
Fig.9 – “7b” area samples extract; milligrams of radical scavenging
activity in 1 g of dry material. Rutin equivalent mg/g dry material.

19. 18
and between the roots samples extract. Only the roots of the sample extract n°2 of the “7b” area has
a slight lower radical scavenging activity compared to the other roots samples of the same area, but
this difference is only about 20mg/g dry material (rutin equivalent).
5. Conclusions
The aim of this study was to analyse and compare the amounts of phenolic compounds,
flavonoids and radical scavenging activity of the aerial parts and roots of Vaccinium myrtillus L.
grown in two different forest conditions (5b: clear cutting 2017; 7b: clear cutting 2015) and sampled
during three different vegetation periods (early May, late May and early August).
1) This study has shown that the two different areas of the forest did not influence the
concentration of phenolic compounds of the samples extract. Same results were obtained for the three
different vegetation periods. The only difference that was obtained was in the sample n°6 from the
“5b” area (early August) that, compared to the other roots samples extract, it had a slight high
concentration of phenolic compounds. This little difference must be attributed at the various
microclimate present in the forest itself.
2) Regarding the concentrations of flavonoids, also in this case the results did not showed
differences between the samples grown in the two different forest areas and sampled from the three
different vegetation periods. In all the samples extract analysed, the roots lacked the presence of
flavonoids. The aerial parts samples extract showed similar results in the amounts of flavonoids
concentration. Also in this case, the little results differences between the samples extract must be
attributed at the different condition in which the plants grow within the forest.
3) The radical scavenging activity was found to be stronger in the aerial parts samples extract
than in the roots samples extract. There are not high differences between the radical scavenging
activity of the samples extract from the two areas of the forest and from the three different vegetation
periods. The roots of the sample extract n°2 (early May) of the “7b” area has a slight lower radical
scavenging activity compared to the other roots samples extract of the same area. This difference is
of only 20 mg/g dry material (rutin equivalent).
The results show that in this study there is not a remarkable difference between the samples
extract from the different areas of the forest and from the different vegetation periods. Nevertheless,
the chemical composition of plant is influenced by many factors and other researches may show
different results from the ones obtained in this study. Further studies would give a better

20. 19
understanding of the relation between a geographical area, a plant vegetation phase and the chemical
composition of Vaccinium myrtillus L. for a better use of its potentials.
Acknowledgment
I would like to thank Kristina Bimbiraitė-Survilienė for guiding me during all the phases of
this research work. A special thanks to Mantas Dūdėnas for the little suggestions that made the
difference.

21. 20
References
Bujor O.C., Le Bourvellec C., Volf I., Popa V.I., Dufour C. ) - Seasonal variations of the phenolic
constituents in bilberry (Vaccinium myrtillus L.) leaves, stems and fruits, and their antioxidant
activity. Food Chemistry. Volume 213, 15 December, pp. 58-68.
Bukantis A. (1994) - Lietuvos klimatas, vadovėlis aukštosioms mokykloms, pp. 134-135.
Burdulis D., Sarkinas A., Jasutuene I., Stackeviciene E., Nikolajevas L., Janulis V. (2009) –
Comparative study of anthocyanin composition, antimicrobial and antioxidant activity in bilberry
(Vaccinium myrtillus L.) and blueberry (Vaccinium corymbosum L.) fruits. Acta Poloniae
Pharmaceutica - Drug Research, Vol. 66 No. 4, pp. 399-408.
European Medicine Agency (2015) - Assessment report on Vaccinium myrtillus L., fructus.
EMA/HMPC/555161/2013. © European Medicines Agency, 2015, pp. 1-83.
Ferrari M., Medici D. (2008) - Alberi e arbusti. Edagricole, p. 878.
Güder A., Gür M., Engin M.S. (2015) - Antidiabetic and Antioxidant Properties of Bilberry
(Vaccinium myrtillus Linn.) Fruit and Their Chemical Composition. J. Agr. Sci. Tech. Vol. 17, pp.
401-414
Jakoola L. (2003) - Flavonoid biosynthesis in bilberry (Vaccinium myrtillus L.). Department of
Biology, University of Oulu. Oulu, Finland, Academic Dissertation, pp. 13-34.
Kaškonienė V., Maruška A., Kornyšova O., Charczun N., Ligor M., Buszewski B. (2009) -
Quantitative and qualitative determination of phenolic compounds in honey. Chemine Technologija
3, pp. 74–80.
Mikašauskaitė J., Ragažinskienė O., Maruška A. (2013) - Variation of total amount of phenolic
compounds, radical scavenging activity and volatile compounds of Liriodendron tulipifera L. and
Ginkgo biloba L. leaves extracts during different vegetation periods. Biologija 59(2), pp. 175–186.
Nestby R., Percival D., Martinussen I., Opstad N., Rohloff J. (2011) - The European Blueberry
(Vaccinium myrtillus L.) and Potential for cultivation. A Review. The European Journal of Plant
Science and Biotechnology 5 (Special Issue I), pp. 5-16.
Nohynek l.J., Alakomi H.L., Kähkönen M.P., Heinonen M., Helander I.M., Oksman-Caldentey
K.M., Puupponen-Pimiä R.H. (2006) - Berry Phenolics: Antimicrobial Properties and Mechanisms
of Action Against Severe Human Pathogens. Nutrition and Cancer, 54(1), pp. 18–32.

22. 21
Poiana M.A., Alexa E., Mateescu C. (2012) - Tracking antioxidant properties and colour changes
in low-sugar bilberry jam as effect of processing, storage and pectin concentration. Chemistry Central
Journal 6:4, pp. 1-14.
Sen CK, Packer L, Hanninen O. (2000) - Handbook of oxidants and antioxidants in exercise. 1st
edt. Elsevier Science B.V. p. 321.
Suzuki R., Tanaka M., Takanashi M., Hussain A., Yuan B., Toyoda H., Kuroda M. (2011) -
Anthocyanidins-enriched bilberry extracts inhibit 3T3-L1 adipocyte differentiation via the insulin
pathway. Nutrition & Metabolism, 8:14, pp. 1-9.
Takikawa M., Inoue S., Horio F., Tsuda T. (2010) - Dietary Anthocyanin-Rich Bilberry Extract
Ameliorates Hyperglycemia and Insulin Sensitivity via Activation of AMP-Activated Protein Kinase
in Diabetic Mice. The Journal of Nutrition. January 20, pp. 527-533.
Uleberg E., Martinussen I., Opstad N., Krogstad T., Rohloff J., Nes A., Nestby R. (2012) -
Cultivation trials of the European blueberry (Vaccinium myrtillus). Conference: X International
Symposium on Vaccinium and Other Superfruits, At Maastricht, The Netherlands. p. 1-3.
Valentovà K., Ulrichovà J., Cvak L., Šimánek V., (2007) – Cytoprotective effect of a bilberry
extract against oxidative damage of rat hepatocytes. Food Chemistry 101, pp. 912-917.
Yamaki K., Kobori M. (2003) - Induction of Apoptosis in Cancer Cells by Bilberry (Vaccinium
myrtillus) and the Anthocyanins. J. Agric. Food Chem., 51 (1), pp 68–75.