Content of Heavy Metals in Mulberry Fruits and Their Extracts-Correlation Analysis

doi:10.4236/ajac.2013.411081

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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

Email: *[email protected]

Received September 29, 2013; revised November 1, 2013; accepted November 12, 2013

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)

(µg·100g−1)

(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

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

Morus

rubra

Morus

nigra

Morus

rubra

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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