American Journal of Analytical Chemistry, 2013, 4, 674-682
Published Online November 2013 (http://www.scirp.org/journal/ajac)
http://dx.doi.org/10.4236/ajac.2013.411081
Open Access AJAC
Content of Heavy Metals in Mulberry Fruits and Their
Extracts-Correlation Analysis
Ružica J. Micić1*, Danica S. Dimitrijević2, Danijela A. Kostić2, Gordana S. Stojanović2,
Snežana S. Mitić2, Milan N. Mitić2, Aleksandra N. Pavlović2, Saša S. Ranđelović2
1Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Pristina, Kosovska Mitrovica, Serbia
2Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Niš, Niš, Serbia
Received September 29, 2013; revised November 1, 2013; accepted November 12, 2013
Copyright © 2013 Ružica J. Micić et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The aim of this research was the determination of heavy metals’ (iron, copper, zinc, manganese, cadmium, nickel and
lead) contents of white, red and black mulberry fruit grown in southeast region of Serbia, and their extracts. The content
of all metals was confirmed in the fruits. All of tree types of mulberry contained the highest content of iron in the fruit
and the lowest content of Cd. The level of iron in all assayed fruits species was ranged between 23.06 - 57.38
mg· 1 0 0g −1. The concentration range of cadmium for the tested fruits was found to be from 1.77 to 2.46 µg·100g−1 in
fruit of Morus nigra L. and Morus alba L., respectively. The content of metals in the fruit of white mulberry decreases
in the following order: Fe > Mn > Zn > Cu > Ni > Pb > Cd. The content of metals in the fruit of red mulberry by de-
creasing the value is: Fe > Zn > Mn > Cu > Ni > Pb > Cd and in the fruit of black mulberry decreases in the following
order: Fe > Zn > Cu > Mn > Ni > Pb > Cd. In addition, an analysis of mulberries extracts showed a significant transfer
of heavy metals during extraction procedure; therefore, the corresponding extraction coefficients reached values up to
73.09%. Those were especially high in the acetone-based extracts. The lowest extraction coefficients were in the
methanol-water extract of red mulberry (0.54%). Moreover, it was established that such coefficients mostly depend on
the solvent nature and also on the treated mulberry species. We found lead in none of the extracts. Pattern recognition
technique, such as principal component analysis (PCA), has been applied to the obtained data with classification and
interdependences among determined metals purposes.
Keywords: Mulberry; Extracts; Heavy Metal; Extraction Coefficient
1. Introduction
The mulberry belongs to the genus Morus of the family
Moraceae. Although it is extensively grown as food for
silkworms in many countries, mulberry fruit production
is a main aim in Turkey, which is one of the most impor-
tant mulberry fruit producers in the world [1,2]. Mul-
berry is found from temperate to subtropical regions of
the northern hemisphere to the tropics of the southern
hemisphere and they can grow in a wide range of cli-
matic, topographical and soil conditions. These are widely
spread throughout all regions from the tropics to the sub-
artic areas. Genus Morus is widespread in Asia, Europe,
North and South America and Africa as well. Mulberry
has a unique delicious fruit, sour and refreshing taste. It
has been used as a folk remedy to treat oral and dental
diseases, diabetes, hypertension, arthritis and anemia [3].
The bright black and purple mulberry fruits, which have
a very pleasant taste when eaten fresh, are also used in
jams, juices, liquors, natural dyes as well as in the cos-
metics industry [4]. Morus species are deciduous and in a
period of low temperatures during the winter are required
to break dormancy. Mulberry fruits may be coloured
white, red or black when they are ripe. Deep-coloured
fruits are good sources of phenolics, including flavonoids,
anthocyanins and carotenoids [5-8], and mulberries are
rich in phenolics [9]. Mulberry has a unique delicious
fruit, sour and refreshing taste. It has been used as a folk
remedy to treat oral and dental diseases, diabetes, hyper-
tension, arthritis and anemia [3].
Fruits and their extracts deserve special attention be-
cause of the important influence they have on human
health. For the majority of the world population, fruits
*Corresponding author.

R. J. MICIĆ ET AL. 675
represent the primary source of the health care. Although
the effectiveness of fruits is mainly associated with their
constituents such as essential oils, vitamins, glycosides,
etc., it was found that prolonged intake can cause health
problems due to the possible presence of heavy metals
[10].
Fruits can easily be contaminated by heavy metals in
the course of cultivation or later during the processing
stage and therefore determining the content of the heavy
metals accumulated is of high importance. The human
body requires both the metallic and the non-metallic
elements within certain permissible limits for growth and
good health. Therefore, the determination of elemental
compositions in food and related products is essential for
understanding their nutritive importance. Accordingly,
the presence of some heavy metals in large quantities in
the body may have a toxic effect [10-13]. The content of
heavy metals is one of the criteria for the use of plant
material in the production of traditional medicines and
herbal infusions.
Rapid and unorganized urbanization and industrializa-
tion have elevated the levels of heavy metals in the envi-
ronment of developing countries [12]. Industrial uses of
metals and other domestic processes have introduced
substantial amounts of potentially toxic heavy metals into
the atmosphere and into aquatic and terrestrial environ-
ments [14]. Heavy metals have a significant toxicity for
human, animals, microorganisms and plants [15]. Thus,
the contaminations of fruit with heavy metals pose a se-
rious threat to its quality and jeopardize food safety [12].
Lead and cadmium are very harmful elements for human
body especially in high concentration [16]. Therefore,
FAO/WHO established the permissible maximum limit
of Cd in fruiting vegetables as 0.05 mg·kg−1 and Pb in
berries and other small fruits as 0.2 mg·kg−1 [17]. Re-
cently, pollution in developing areas has been increasing.
Various factors such as exhaust gas, industrial waste and
waste water have increased the heavy metal contamina-
tion in fruit and other edible parts of plants. The most
important cause of pollution for the plants on roadsides is
probably exhaustion of gases [18].
The aim of this investigation was to determine the con-
centrations of heavy metals in the mulberries and their
extracts and to determine the coefficient of extraction of
metals in different solvents and their mixtures.
2. Experimental
2.1. Samples
Fruits of mulberries were collected in the South East
Serbia in early July 2011. Fruit maturity was estimated
on the basis of the color that was very black. Samples
were stored in plastic bags and kept frozen until extrac-
tion. The study area is located in the surroundings of the
city of Niš. Niš has about 300.000 inhabitants and it is
the third-largest city in the country after Belgrade and
Novi Sad; however, the industry in this area is poorly
developed.
2.2. Reagents
All the reagents used were of the analytical purity
(Merck, Germany). The working solutions were prepared
immediately before the analysis from the basic solution
with 1000 mg·l−1 concentration for all metals. For the
preparation of standard solutions high purity Milli-Q
water was used. The glassware and polyethylene con-
tainers used for analysis were washed with tap water,
then soaked over the night in 6 M HNO3 solution and
rinsed several times with ultra pure water to eliminate
absorbance due to detergent.
2.3. Apparatus
Atomic absorption measurements were made using a
Varian SpectraAA 10 with background correction and
hollow cathode lamps. Air-acetylene flame was used for
determination of all the elements.
2.4. Procedure
The standard procedure described by Association of Of-
ficial Analytical Chemists (AOAC) was followed for the
preparation of the samples for the analysis of heavy met-
als [19]. Accurately weighed (2 g) sample was trans-
ferred into a silica crucible and kept in a muffle furnace
for ashing at 450˚C for 3 h and then 5 ml of 6 M hydro-
chloric acid was added to the crucible. Care was taken to
ensure that all the ash came into contact with acid. Fur-
ther, the crucible containing acid solution was kept on a
hot plate and digested to obtain a clean solution. The
final residue was dissolved in 0.1 M nitric acid solution
and made up to 50 ml. Working standard solutions were
prepared by diluting the stock solution with 0.1 M nitric
acid for checking the linearity.
Black, red and white mulberry fresh fruits (10 g) was
extracted with water, ethanol-water (50/50, v/v%), ethanol,
acetone-water (50/50, v/v%), acetone, methanol-water
(50/50, v/v%) and methanol. All solvents were acidified
with 1 ml concentrated HCl. The extraction was per-
formed with 100 ml of solvents using the ultrasonic bath
for 30 minutes. The suspension was gravity filtered
through a Buchner funnel and Whatman No. 1 filter pa-
per. Extracts were stored in the fridge until their ana-
lysis.
2.5. Extraction Coefficient
Extraction coefficient, EC, is defined by the Equation
Open Access AJAC

R. J. MICIĆ ET AL.
Open Access AJAC
676
3.1. Content of Heavy Metals in Mulberry Fruits (1):

Mextract Mfruit
EC100 CC

(1) The highest content of metal in the mulberries fruits was
iron and the lowest content was Cd. The level of iron in
all assayed fruits species was ranged between (23.06 -
57.38 mg·100g−1) (Table 1). Fruit materials from all ori-
gins accumulated iron above the limit proposed by FAO/
WHO in edible plants (2 mg·100g−1) [20]. However, the
results of this study are well comparable with the data
reported earlier for iron content of other authors.
where CMextract is content of the metal in the extract and
CMfruit is content of the metal in the fruit.
2.6. Statistical Analysis
Data are presented as the mean ± standard deviation (SD)
for triplicate determinations. All statistical calculations
were made using Statistica package (Statistica 8.0,
StatSoft, Inc., Tulsa, OK, USA).
The concentration range of copper for the assayed
fruits found to be (0.86 - 1.51 mg·100g−1) (Table 1). The
results obtained for all the plant materials are in the per-
missible limit of copper set by China (2 mg·100g−1) for
medicinal plants [21]. The concentration of copper ob-
tained in the fruits of the present study is comparable
with the reported values for other authors.
3. Results and Discussion
In our work the content of heavy elements, iron (Fe),
zinc (Zn), copper (Cu), manganese (Mn), nickel (Ni),
lead (Pb), and cadmium (Cd), was determined in the
mulberries fruits from the region of Southeast Serbia and
their extracts (Table 1). The content of all metals was
confirmed.
Manganese content in the mulberries fruits was ranged
from 0.81 mg·100g−1 in Morus nigra L. fruit to 2.33
mg· 1 0 0g −1 in Morus alba L. fruit.
Table 1. The content of heavy metals in white, red and black mulberry and their extracts.
Sample
Solvent Fe
(mg·100g−1)
Cu
(mg·100g−1)
Mn
(mg·100g−1)
Cd
(µg·100g−1)
Ni
(mg·100g−1)
Zn
(mg·100g−1)
Pb
(mg·100g−1)
Fruit 23.06 ± 1.02 0.86 ± 3.11 2.33 ± 0.18 2.46 ± 0.21 0.36 ± 0.01 2.23 ± 0.17 0.09 ± 0.01
Water 0.32 ± 0.01 0.08 ± 0.02 0.28 ± 0.02 - 0.10 ± 0.01 0.73 ± 0.05 -
Ethanol/water 0.15 ± 0.16 0.04 ± 0.02 0.27 ± 0.01 - 0.05 ± 0.01 0.59 ± 0.04 -
Ethanol 0.71 ± 0.14 0.08 ± 1.36 0.44 ± 0.03 0.33 ± 0.02 0.04 ± 0.01 0.86 ± 0.07 -
Acetone/water 1.44 ± 0.04 0.08 ± 0.01 0.42 ± 0.04 0.42 ± 0.03 0.05 ± 0.01 1.05 ± 0.08 -
Acetone 5.38 ± 4.11 0.26 ± 0.19 0.59 ± 0.04 0.25 ± 0.02 0.03 ± 0.01 1.63 ± 0.23 -
Methanol/water 0.35 ± 0.30 0.04 ± 0.54 0.15 ± 0.01 - 0.14 ± 0.02 0.91 ± 0.07 -
Morus alba L.
Methanol 0.23 ± 0.18 0.11 ± 0.03 0.46 ± 0.01 - 0.10 ± 0.01 1.12 ± 0.08 -
Fruit 57.38 ± 6.25 1.51 ± 0.12 1.98 ± 0.20 1.84 ± 0.15 0.37 ± 0.03 5.04 ± 0.06 0.20 ± 0.02
Water 1.05 ± 0.78 0.01 ± 0.19 0.09 ± 0.01 - 0.05 ± 0.01 0.43 ± 0.03 -
Ethanol/water 0.70 ± 0.10 0.02 ± 0.00 0.15 ± 0.01 - 0.04 ± 0.00 0.43 ± 0.03 -
Ethanol 0.32 ± 0.94 0.01 ± 0.00 0.18 ± 0.01 - 0.02 ± 0.00 0.57 ± 0.02 -
Acetone/water 5.85 ± 1.16 0.05 ± 0.01 0.39 ± 0.02 0.29 ± 0.02 0.06 ± 0.01 0.55 ± 0.02 -
Acetone 5.69 ± 0.36 0.28 ± 0.01 0.59 ± 0.04 0.27 ± 0.04 0.05 ± 0.01 1.04 ± 0.02 -
Methanol/water 0.31 ± 5.10 0.09 ± 0.01 0.09 ± 0.01 - 0.04 ± 0.00 0.08 ± 0.01 -
Morus rubra L.
Methanol 0.32 ± 0.09 0.11 ± 0.01 0.25 ± 0.02 - 0.02 ± 0.00 0.12 ± 0.01 -
Fruit 42.13 ± 8.14 1.07 ± 0.11 0.81 ± 0.06 1.77 ± 0.13 0.27 ± 0.00 3.40 ± 0.32 0.14 ± 0.01
Water 0.23 ± 1.96 0.02 ± 0.01 0.07 ± 0.01 - 0.05 ± 0.01 0.04 ± 0.01 -
Ethanol/water 0.70 ± 0.02 0.02 ± 0.00 0.10 ± 0.01 - 0.03 ± 0.00 0.08 ± 0.01 -
Ethanol 0.50 ± 0.00 0.01 ± 0.00 0.20 ± 0.02 0.34 ± 0.02 0.02 ± 0.00 0.91 ± 0.05 -
Acetone/water 12.62 ± 3.68 0.01 ± 0.0 0.41 ± 0.05 0.85 ± 0.07 0.01 ± 0.00 1.56 ± 0.04 -
Acetone 5.28 ± 0.03 0.28 ± 0.03 0.54 ± 0.03 0.26 ± 0.01 0.03 ± 0.00 1.50 ± 0.00 -
Methanol/water 0.80 ± 1.01 0.06 ± 0.01 0.08 ± 0.01 - 0.05 ± 0.01 0.99 ± 1.00 -
Morus nigra L.
Methanol 0.48 ± 3.01 0.08 ± 0.01 0.19 ± 0.02 0.18 ± 0.01 0.03 ± 0.01 0.55 ± 3.12 -

R. J. MICIĆ ET AL. 677
The concentration range of cadmium for the tested
fruits was found to be from 1.77 to 2.46 µg·100g−1 in
fruit of Morus nigra L. and Morus alba L., respectively.
Cadmium accumulates in human body and damages
mainly the kidneys and liver [21]. For medicinal plants
the permissible limit set by WHO, China and Thailand
was 0.03 mg·100g−1 cadmium in finished herbal products
[13]. All the investigated plant materials accumulated
above the permissible limit.
The content of nickel was similar in all three types of
fruits (0.36, 0.37 and 0.27 mg·100g−1 for white, red and
black mulberry fruit, respectively).
The highest content of zinc was in the fruit of Morus
rubra L. (5.04 mg·100g−1) and the lowest in the fruit of
Morus alba L. (2.23 mg·100g−1). Zinc content of the
studied medicinal plants has been compared with the
limit proposed by FAO/WHO in edible plants (2.74
mg· 1 0 0g −1) [20], it was found that only Morus alba L.
within this limit while all other plants accumulated zinc
above this limit. However, for medicinal plants the WHO
limits have not yet been established for zinc [13].
Lead content in the mulberries fruits ranged from 0.09
mg· 1 0 0g −1 in Morus alba L. fruit to 0.2 mg·100g−1 in
Morus rubra L. fruit (Table 1). The content of lead in the
investigated medicinal plants is under the permissible
limit for medicinal plants set by China, Malaysia, Thai-
land and WHO (1 mg·100g−1) [13]. Lead content con-
firmed only in the fruits of mulberries but not in their
extracts. Trichopoulos [22] has reported the Pb has a
toxic effect for human metabolism even in low amounts
and may have carcinogenic effects.
All of three types of mulberry had the highest content
of Fe in the fruit and the lowest content of Cd. The con-
tent of metals in the fruit of white mulberry decreases in
the following order: Fe > Mn > Zn > Cu > Ni > Pb > Cd.
The content of metals in the fruit of red mulberry by de-
creasing the value is: Fe > Zn > Mn > Cu > Ni > Pb > Cd.
The content of metals in the fruit of black mulberry de-
creases in the following order: Fe > Zn > Cu > Mn > Ni
> Pb > Cd (Table 1).
3.2. Content of Heavy Metals in Mulberry
Extracts
In the extracts, the highest concentration of iron was in
acetone-water extract (50/50, v/v%) of Morus nigra L.
(12.62 mg·100g−1) and the lowest concentration was in
ethanol-water extracts (50/50, v/v%) of Morus alba L.
(0.15 mg·100g−1). The content of copper in the extracts
was ranged from 0.01 mg·100g−1 in water and ethanol-
water (50/50, v/v%) of Morus rubra L. and in ethanol
and acetone-water of Morus nigra L. to 0.28 mg·100g−1
in acetone extract of Morus rub ra L. and Morus nigra L.
Manganese content was the highest in acetone extracts of
Morus alba L. and Morus rubra L. (0.59 mg·100g−1) and
the lowest concentration was in the methanol-water ex-
tract of Morus nigra L. (0.08 mg·100g−1). The content of
cadmium was ranged from 0.18 µg·100g−1 in the Morus
nigra L. methanol extract to 0.85 µg·100g−1 in the ace-
tone-water (50/50, v/v%) of Morus nigra L. All the water,
ethanol-water and methanol-water extracts of fruits, and
methanol extracts of white and red mulberry did not con-
firm the content of Cd. Nickel content was low and
ranged from 0.01 mg·100g−1 in acetone-water (50/50,
v/v%) of Morus nigra L. to 0.14 mg·100g−1 in methanol-
water extract of Morus alba L. The highest content of
zinc was in acetone extract of Morus alba L. (1.63
mg· 1 0 0g −1) and the lowest content was in water extract
of Morus nigra L. (0.04 mg·100g−1). The content of lead
was not confirmed in the extracts (Table 1).
The highest content of Fe, Cu, Mn and Zn of Morus
alba L. fruit showed the acetone extract (5.38, 0.26, 0.59
and 1.63 mg·100g−1, respectively). The highest content of
Cd in white mulberry extracts was in the acetone-water
extract (0.42 µg·100g−1), while water, ethanol-water,
methanol and methanol-water did not confirm the content
of Cd. The methanol-water extract of Morus alba L. fruit
showed the highest content of Ni (0.14 mg·100g−1). The
ethanol-water extract of Morus alba L. fruit had the low-
est content of Fe, Cu and Zn (0.15, 0.04 and 0.59
mg· 1 0 0g −1, respectively). The lowest content of Mn was
in the methanol-water extract of white mulberry fruit
(0.15 mg·100g−1). The acetone extract showed the lowest
content of Ni (0.03 mg·100g−1).
The highest contents of metals in Morus rubra L. ex-
tracts showed the acetone and acetone-water extracts.
The acetone-water extract had the highest content of Fe,
Cd and Ni (5.85, 0.29 and 0.06 mg·100g−1, respectively).
The highest content of Cu, Mn and Zn was in the acetone
extract of Morus rubra L. fruit (0.28, 0.59 and 1.04
mg· 1 0 0g −1, respectively). The water, ethanol-water, etha-
nol, methanol-water and methanol extracts of red mul-
berry fruit did not confirm the content of Cd. The lowest
content of Fe, Mn and Zn was in the methanol-water ex-
tract of Morus rubra L. fruit (0.31, 0.09 and 0.08
mg· 1 0 0g −1, respectively). The water and ethanol extract
of red mulberry fruit showed the lowest content of Cu
(0.01 mg·100g−1). The ethanol extract had the lowest
content of Ni (0.02 mg·100g−1). The manganese content
was the lowest in water and methanol-water extract of
red mulberry (0.09 mg·100g−1).
The highest content of Fe in Morus nigra L. extracts
was in acetone-water extract. Also, the acetone-water
extract showed the highest content of Cd and Zn. The
content of Cu and Mn was the highest in the acetone ex-
tract. The water and methanol-water extracts of Morus
nigra L. fruit showed the highest content of Ni. The wa-
ter, ethanol-water and methanol-water extracts of black
mulberry fruit did not confirm the content of Cd (Table
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R. J. MICIĆ ET AL. 678
1). The water extract of black mulberry fruit had the
lowest content of Fe, Mn and Zn (0.23, 0.07 and 0.04
mg· 1 0 0g −1, respectively). The lowest content of Cu and
Ni was in the acetone-water extract (0.01 mg·100g−1).
3.3. Coefficient of Extraction
Based on the results, the analyzed elements can be clas-
sified into three groups: elements with the low extraction
coefficient (less than 10%); elements with the medium
extraction coefficient (10% - 30%), and elements with
the high extraction coefficient (more than 30%).
The extraction coefficients EC obtained in this study
varied markedly, from 0% to 73.06% (Table 2, Figures
1-3).
The extraction coefficient depends mostly on the ex-
traction medium. The lowest transfer of heavy metals
was in the methanol-water extract (50/50, v/v%) in
Morus rubra L. (0.54%) for extraction of Fe, and the
highest in the acetone extract of Morus alba L. (73.09%)
for extraction of Zn. The extraction coefficient also de-
pends on the plant species that is being extracted (Table
2).
The water extracts all of three types of mulberry had
Table 2. Extraction coefficient of heavy metals from the
mulberries by different extractions.
EC (%)
Sample Solvent
Fe CuMn Cd Ni Zn
Water 1.38 9.30 12.02 - 27.7832.74
Ethanol/water 0.65 4.65 11.59 - 13.8926.46
Ethanol 3.08 9.30 18.88 13.41 11.1138.57
Acetone/water 6.24 9.30 18.03 17.07 13.8947.09
Acetone 23.33 30.23 25.32 10.16 8.3373.09
Methanol/water 1.52 4.65 6.44 - 38.8940.81
Morus
alba L.
Methanol 1.00 12.80 19.74 - 27.7850.22
Water 1.83 0.66 4.55 - 13.518.53
Ethanol/water 1.22 1.32 7.58 - 10.818.53
Ethanol 0.56 0.66 9.09 - 5.4111.31
Acetone/water 10.20 3.31 19.70 15.76 16.2210.91
Acetone 9.92 18.54 29.80 14.67 13.51 20.63
Methanol/water 0.54 5.96 4.55 - 10.811.59
Morus
rubra L.
Methanol 0.56 7.28 12.63 - 5.412.38
Water 0.55 1.87 8.64 - 18.521.18
Ethanol/water 1.66 1.87 12.35 - 11.112.35
Ethanol 1.19 0.93 24.69 19.21 7.41 26.76
Acetone/water 30.00 0.93 50.62 48.02 3.70 45.88
Acetone 12.53 26.17 66.67 14.69 11.11 44.12
Methanol/water 1.90 5.61 9.88 - 18.5229.12
Morus
nigra L.
Methanol 1.14 7.48 23.46 10.17 11.1116.18
Figure 1. Extraction coefficient in extracts of Morus alba L.
Figure 2. Extraction coefficient in extracts of Morus rubra L.
Figure 3. Extraction coefficient in extracts of Morus nigra L.
relatively low coefficients of extraction except for ex-
traction of Zn in Morus alba L. (32.74%). The ethanol-
water and ethanol extracts showed the lowest coefficients
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R. J. MICIĆ ET AL. 679
of extraction. The ethanol-water extract showed the high-
est coefficient of extraction for extraction of Zn in Morus
alba L. (26.46%). The lowest coefficient of extraction
was for extraction of Fe (0.65%). The coefficient of ex-
traction was the highest in the acetone and acetone-water
extracts all of three types of mulberry. The acetone-water
extracts had a high coefficient of extraction in Morus
alba L. for extraction of Zn and in Morus nigra L. for
extraction of Fe, Mn, Cd and Zn. In the other cases it was
lower than 30%. The acetone extract of Morus alba L.
fruit showed the highest coefficient of extraction for ex-
traction of Zn (73.09%). A high coefficient of extraction
(more then 30%) was for extraction of Cu in white mul-
berry (30.23%), Mn and Zn in black mulberry (66.67%
and 44.12%, respectively). The methanol-water extracts
of white, red and black mulberry had a coefficient of
extraction less than 30% except for extraction Ni and Zn
in Morus alba L. fruit extract (38.89% and 40.81%, re-
spectively). Also, the methanol-water extract showed the
lowest coefficient of extraction, in relate of all extracts,
for extraction of Fe in Morus rubra L. fruit extract. The
methanol extract showed a high and the highest coeffi-
cient of extraction in Morus alba L. fruit extract for ex-
traction of Zn (50.22%) (Table 2).
From the obtained results of elemental concentrations
in investigated plant samples after using different extrac-
tion solvents, it can be concluded that there is a sifnifi-
cant difference (p < 0.05) in elemental profiles among
plant samples.
3.4. PCA Analysis
I order to highlight the relation between the elements the
principal component analysis (PCA) was used. With
PCA, the data reduction is performed by transforming the
data into orthogonal components that are a linear combi-
nation of the origin variables. One of the main objectives
of PCA is to identify factors that are substantially mean-
ingful. The principal components which have eigenval-
ues higher than 1 were extracted (Kaiser criterion) [23,24]
(Figure 4).
This led to the formation of two principal components.
The first component accounted for 55.64% and the sec-
ond for 21.96% of the total variation of data. The first
two components account for 77.60% of variances for all
of the data. The first component represents the maximum
variation of the data set.
Factor loading and communality for each element is
presented in Table 3.
Factor loadings of elements data from Table 3 indicate
the similarities and correlations between elements. The
elements with small factor loadings have only little in-
fluence on data structure, whereas the elements with high
loadings represent those elements with the greatest in-
fluence on the grouping and separation of plant samples.
Figure 4. Eigenvalues of the correlation matrix.
Table 3. The loadings and the scores of all extracted factors.
Factor 1
(PC1)
Factor 2
(PC2)Factor 3 Factor 4 Factor 5Factor 6
Fe −0.8437*−0.30550.1789 0.3599 0.1681−0.0712
Cu −0.8088*−0.4549−0.5201 0.1033 0.08350.1406
Mn −0.8826*0.3244 −0.1152 −0.0094 −0.2837 −0.1478
Cd −0.4338 0.6389*0.2694 −0.0497 −0.18070.1768
Ni 0.25610.7558*0.5811 0.1448 −0.0448 0.0508
Zn −0.8523*0.22450.2479 −0.3327 0.2233 −0.0335
Eigenvalue3.34 1.32 0.79 0.27 0.20 0.08
Variance
(%) 55.6421.9613.12 4.57 3.34 1.36
Cumulative
(%) 55.6477.60 90.73 95.30 98.64100
*Loadings >0.5 and <−0.5.
The first factor with 55.64% of variance comprises Fe,
Mn, Cu and Zn with high loadings and Ni with low load-
ings. All elements, except Ni have negative loadings in
this factor. A negative correlation supports the fact that
there is a strong influence between matrix elements and
traces. No significant loading value, except for Cd and Ni,
was obtained for any variable of the second factor, which
is responsible for 21.96% of total variance. This factor,
dominatly loaded by Ni, refers to potential anthropogenic
contaminant [25].
Correlation analysis of total metal contents (Table 4)
in different plant extracts is also showed strong correla-
tions in two groups of elements. In general, interpretation
of correlation analysis were done using correlation coef-
ficient values higher than 0.5 [26].
The correlation coefficients can range from −1 to +1
and is independent of the units of measurement. Inter-
pretation of correlation analysis enabled two groupings to
be obtained. A close relation was observed between the
concentrations of Fe, Mn, Cu and Zn while there is not a
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R. J. MICIĆ ET AL. 680
Table 4. Correlation matrix for the element concentrations
in plant samples.
Fe Cu Mn Cd Ni Zn
Fe 1.0000 0.8087* 0.5844*0.3182 −0.3021 0.6150*
Cu 1.000 0.7487*0.1320 −0.0389 0.5118*
Mn 1.00000.4608 −0.0439 0.7413*
Cd 1.0000 −0.3845 0.4243
Ni 1.0000 0.0356
Zn 1.0000
*Marked correlations are significant at p < 0.05.
significant correlation of these metals with Cd and Ni.
3.5. Heavy Metals in the Mulberry Fruits from
Different Regions
The comparison of our results regarding the heavy metals
content in the mulberries from the region of Southeast
Serbia with the results of other authors is shown in Table
5.
Several papers were previously published on the com-
position of micro-, macro- and toxic elements in various
mulberry species [2,27,28]. Those studies were mainly
focused on the investigation of the mineral fruit con-
stituents of a range of the mulberry species, and showed
that mulberry fruit contains an essential macro-elements
as potassium (K), calcium (Ca), magnesium (Mg) and
sodium (Na) and micro-elements as iron (Fe), zinc (Zn)
and nickel (Ni) as indicated in Table 3. Investigation of
constituents in various mulberry species performed in
different countries, gave the results of Ca content from
132 to 574 mg·100g−1 in fresh fruit, Mg from 91 to 240
mg· 1 0 0g −1 and Na from 45 to 280 mg·100g−1. Trace ele-
ments in these tested extracts of mulberry fruit were in
the range from 3 to 77.6 mg·100g−1, with Fe and Zn be-
ing the most dominant elements and Cu the least domi-
nant element in relation to all detected elements. Suffi-
cient quantities of essential macro- (K, Ca, Mg, and Na)
and micro- (Fe, Zn, and Ni) elements were found in all
the fruits. Potassium was the predominant element with
concentration ranging from 834 to 1731 mg·100g−1. The
decreasing order of micro-minerals was Fe > Zn > Ni.
Nitrogen (N), phosphorus (P), copper (Cu) and manga-
nese (Mn) were not determined [27] (Table 5). Fruit of
Morus alba L. from Turkey [28] has a lower content of
heavy metals than our results (Table 5).
Preliminary study of heavy metals in mulberry fruits
from Southeast region of Serbia and their extracts show
that the iron content was the highest (13.8 - 42.3
mg· 1 0 0g −1) while the contents of Cu, Zn and Mn were
remarkably lower, and ranged from 0.9 to 6.2 mg·100g−1
which is less than content of heavy metals in mulberries
from Southeast Serbia [29].
Table 5. Contents of mineral element from mulberry fruit
from different countries.
Morus
nigra
L.
Morus
rubra
L.
Morus
nigra
L.
Morus
rubra
L.
Morus
alba L. Morus nigra L.
Morus
alba L.
(mg·100g−1)
Turkey Turkey Turkey Turkey Turkey PakistanPakistan
N 80069092 82 75 n.d.* n.d.*
P 289242232226 247 n.d.*n.d.*
K 1005929922834 1668 12701731
Ca 137143132132 152 470 574
Mg 10891 106115 106 240 240
Na 58 45 59 61 60 272 280
Fe 5 5 4.2 4.5 4.2 77.6 73
Cu n.d.*n.d.*0.4 0.4 0.5 n.d.*n.d.*
Mn 7 5 4.2 4.0 3.8 n.d.*n.d.*
Ni n.d.*n.d.*n.d.* n.d.* n.d.* 1.6 2.2
Zn 3 3 3.2 3.2 2.8 59.2 50.2
References[27][27][2] [2] [2] [28] [28]
*not determined.
In different environments, many factors are known to
affect the concentration of heavy metals in both soil and
plants, including industrialization, traffic density and
unknown atmospheric deposits. In the mulberries from
the region of Southeast Serbia, the highest content is that
of Fe, while the content of Cd is significantly lower. This
can be attributed, among other factors, to the fact that
these were mulberries growing in a non-polluted region
of Southeast Serbia.
4. Conclusions
The analysis of white, red and black mulberry fruit and
their extracts from the region of Southeast Serbia showed
the presence of Fe, Cu, Mn, Cd, Ni, Zn and Pb. The iron
concentration in the fruits was rather high while the cad-
mium content was significantly lower. The extracts of
fruits did not confirm the lead content.
By analyzing the extracts of mulberries, the transfer of
heavy metals from the plants to the extracts was found.
The extraction coefficients varied in the interval from
0.54% to 73.09%. The extraction coefficient depends
mainly on the extraction medium. The lowest transfer of
heavy metals was found in methanol-water extract, and
the highest in acetone extract. Since the white, red and
black mulberries and their extracts are used in traditional
medicine, there is a possible danger of heavy metal poi-
soning, if they come from the polluted areas. Therefore,
the fruits should be collected in non-polluted regions and
they should be tested for the presence of heavy metals.
These investigations are obligatory and they are recom-
mended by the European standards in order to prevent
poisoning by heavy metals.
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R. J. MICIĆ ET AL. 681
5. Acknowledgements
Financial support of this work by the Serbian Ministry of
Education and Science, Project No. ON 172047.
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